A method for quantitative measurement of a target marker by in situ immunofluorescence comprising the steps of: a) providing a sample immobilized on a sample support; b) carrying out a staining step comprising incubating said sample with at least one target probe, at least one screening probe for imaging the sample region of interest (first screening probe) and optionally at least one further screening probe for the sample region of non-interest to be discarded in further analysis (second screening probe), wherein incubation times of the sample with each of said probes are sufficiently low to avoid saturation of the sample with the probes, while ensuring suitable staining of the sample so that a linear relationship between the target marker concentration and the resultant fluorescent signal intensity can be formed, wherein incubation times of the sample with each of said probes are less than 16 minutes; c) acquiring an image of the stained sample comprising raw signals emitted by each of the imaging probes; d) generating a target evaluation mask comprising an active area for analysis of said target marker, comprising defining a threshold for signals of said at least one screening probe, and assigning binary values to screening probe signals, said binary values comprising a first value (for example 0) and a second value (for example 1), whereby screening probe signal values that are beyond said corresponding threshold are set to said first value, and screening probe signal values that are not beyond said corresponding threshold are set to said second value, said active area of the mask being defined by areas of the image comprising signals set to said second value; e) applying the active area of the target evaluation mask on the raw signal values of the first screening probe to obtain a screening probe data set comprising values proportional to the raw signal values of the first screening probe for the active area; f) processing signals of the target probe, including extracting target probe signal data limited to said active area to obtain a target probe data set comprising target probe signal values for the active area; and g) generating a sample output including combining said target probe data set with said screening probe data set to provide information on quantitative levels of the target marker. a) providing a sample immobilized on a sample support; b) carrying out a staining step comprising incubating said sample with at least one target probe, at least one screening probe for imaging the sample region of interest (first screening probe) and optionally at least one further screening probe for the sample region of non-interest to be discarded in further analysis (second screening probe), wherein incubation times of the sample with each of said probes are sufficiently low to avoid saturation of the sample with the probes, while ensuring suitable staining of the sample so that a linear relationship between the target marker concentration and the resultant fluorescent signal intensity can be formed, wherein incubation times of the sample with each of said probes are less than 16 minutes; c) acquiring an image of the stained sample comprising raw signals emitted by each of the imaging probes; d) generating a target evaluation mask comprising an active area for analysis of said target marker, comprising defining a threshold for signals of said at least one screening probe, and assigning binary values to screening probe signals, said binary values comprising a first value (for example 0) and a second value (for example 1), whereby screening probe signal values that are beyond said corresponding threshold are set to said first value, and screening probe signal values that are not beyond said corresponding threshold are set to said second value, said active area of the mask being defined by areas of the image comprising signals set to said second value; e) applying the active area of the target evaluation mask on the raw signal values of the first screening probe to obtain a screening probe data set comprising values proportional to the raw signal values of the first screening probe for the active area; f) processing signals of the target probe, including extracting target probe signal data limited to said active area to obtain a target probe data set comprising target probe signal values for the active area; and g) generating a sample output including combining said target probe data set with said screening probe data set to provide information on quantitative levels of the target marker.