Stuart joined Charles Sturt's Central Australian Expedition to search for an inland sea in August 1844. Sturt was so sure that there was an inland sea in the centre of Australia, that he took a large boat as part of his equipment. Stuart joined the group as a draftsman, who would draw up the maps. He was paid one pound a week, and provided with food. Stuart and James Poole, the second in command, were sent out ahead of the main group to find water. The main group could only travel as fast as their flock of sheep could walk. Stuart and Poole found water at Depot Glen, near the current site of Milparinka.
In the hot dry climate of summer, the group remained trapped at the waterhole for seven months. Stuart took a small group to find water to the north, west, and east, but found none. Stuart was left behind in charge of the main group at Depot Glen. It was very hot so they dug an underground room to keep cool. All the men became sick from scurvy because of the lack of fresh fruit and vegetables. This caused their gums to become soft and their teeth to fall out. They had headaches, their noses bled, and their skin began to turn black. When Poole died, Stuart was made second in command. He also became the surveyor and did all the mapping as Sturt could not see properly. When it finally rained the group tried to keep going north but were blocked by the sand dunes of the Simpson Desert. They turned south and went back to the Darling River. Stuart took over as leader when Sturt became blind and too ill to lead the group. They arrived back in Adelaide after six weeks hard travelling. Sturt had to be carried in a cart, and Stuart, crippled with scurvy and beriberi, looked like a skeleton.
It took Stuart nearly a year to recover from the expedition. He wrote in a letter: "I lost the power of my limbs for ... six months, and was quite unable to do anything for myself for ... twelve months." For the next few years he was unable to work. The money he had made was spent on doctors and living expenses. In 1849 he moved to near Port Lincoln on the Eyre Peninsula and worked on a farm. He soon found work surveying in the area, and met William Finke and James Chambers. Finke and Chambers were rich men and they paid Stuart to explore for them. They wanted him to find them new farm lands and water, as well as minerals like gold and copper.
Stuart learned from his trip with Sturt that large groups were not able to move quickly through the dry Australian lands. He believed that a small group would have more success. Finke paid him to find out if the land north of Lake Torrens would be suitable for sheep. On this first trip he went with two men, Forster and an unnamed Aboriginal youth. They took six horses, enough food for six weeks, a watch and a compass. In May 1858 they went through settled areas until they reached Oratunga Station, one of the most remote farms in South Australia. It is near the present day town of Blinman, in the north of the Flinders Ranges. From here they went northwest and found a permanent water hole, Andamooka Waterhole, on a creek Stuart named Chambers Creek. It has since been renamed Stuart Creek. The country was very dry, and they could only find water in Aboriginal wells. The stony ground hurt the horses feet. At the end of July they reached the site of the present day town of Coober Pedy. Their horses were in poor condition and they were running out of food. Stuart decided to change direction to the south west and try and reach the coast near Fowlers Bay. The Aboriginal youth would not go any further and went back. He was scared of other Aborigines in the area they were going into. Stuart said the land was "...dreary, dismal, dreadful desert." Stuart and Forster reached the coast at Denial Bay in August 1858, and were back in settled farm lands by 11 September. They had completed a journey of in four months. He gave his diary and maps to the South Australian government. As a reward, Stuart was given the lease to 1,000 square miles of land at Chambers Creek.
The official South Australian government explorer Benjamin Herschel Babbage followed Stuart's maps and went to Chambers Creek. He reported that the map showed the creek was marked too far to the north. Stuart went back to Chambers Creek to survey the area again. This expedition was paid for by James Chambers and William Finke who realized the value of reliable water supplies on the other side of the salt lakes. Stuart left Oratunga Station on 2 April 1859, with a small group, three men and 14 horses. The men he took with him included a Bavarian naturalist and artist, David Herrgott, and Louis Müller, a stockman and botanist. Both Herrgott and Müller had been gold miners in Victoria, and would know how to look for gold in the country around Chambers Creek. They did not carry much equipment, only a blanket to sleep with, but no tents. The only food they took was flour, dried beef (jerky), tea, sugar and tobacco. He took more equipment than just his watch and compass on this trip; a sextant, a copy of the Astronomical Almanac and a telescope.
Stuart went a different way to Chambers Creek, going through a gap in between the salt lakes that had been explored by Major Peter Warburton earlier in the year. Herrgott found a group of 12 artesian springs, which Stuart named after him. This name later became the settlement of Herggott Springs. The name was changed again during World War One because of strong anti-German feelings in Australia. The town is now called Marree after the Aboriginal word for possum. These springs were mound springs; over thousands of years the salt in the water had formed small hills, and the water came out of them like water out of a volcano. Some of the mounds were over high. They were valuable because they provided reliable water for people and animals moving north to Chambers Creek.
After surveying Chambers Creek again, Stuart explored the area to the north west of Chambers Creek. They found more mound springs which Stuart named Elizabeth Springs after one of Chamber's daughters. They continued north west and searched for gold in the Davenport Range. They found more springs which they named the Spring of Hope. They were able to continue and found more springs, which Stuart named Freeling Springs, after Major Freeling, a South Australian politician. By the time they had passed the current site of Oodnadatta, both the men and the horses were suffering from the lack of water and proper food. The horses' shoes had worn out in the rocks around the Davenport Range. On 12 June 1859, Stuart turned around and went back. They rode south back to Port Augusta and then took a boat back to Adelaide.
The South Australian government offered a prize of £2,000 to the first person to cross Australia from the south to the north. They hoped this would be a route for the Australian Overland Telegraph Line that would connect Australia to the line that came from Europe. Stuart and Chambers plans for the trip were not accepted by the government. The government sent Alexander Tolmer to lead the trip. His expedition did not make it out of the settled areas. In August 1859, one month after his last trip, Stuart went back to do more surveying for Chambers at Chambers Creek. With William Darton Kekwick and two other men, they took 12 horses and explored the west side of Lake Eyre. Chambers and Stuart believed that the lake might hold water. Instead Stuart found mud after he walked several kilometres into the dry salt lake. He found another artesian mound spring, more than in height, which he named William Springs, after one of Chamber's sons. He then set off for the Spring of Hope. He saw grasslands and eventually made a base at Freeling Springs. He explored much of the surrounding area hoping to find gold. On 6 January 1860, with his food supplies getting low, and two of the men refusing to go any further, Stuart once again returned home.