A method for focusing an image of a slide (1, 1') provided with a matrix and one or more pits (10, 10') in a fluorescent test for autoimmunity through an optical microscope provided with at least two lenses and a camera to acquire images at two different magnifications and a movable table on which is placed the slide, the matrix being the material outside the pit, and comprising the operations of: - Selection of a lens at a first magnification (B.I.) and determination of a first focus distance (Z-foc-BI) at said first magnification (B.I.) for at least one pit (10, 10') of the slide (1, 1'), said focusing operation comprising the acquisition of images at different incremental distances (Δ) at a point (Po) predetermined of the matrix of the slide located outside of the pit and the focus analysis of said acquired images; - Subdivision of the pit under examination in a plurality of points (200', 200) defining an acquisition path of images; - Selection of a lens at a second magnification (A.I.) greater than the first magnification (B.I.); - For each point (200', 200) determination of a punctual focus distance (Z-foc-punt) at said second magnification (A.I.) through the acquisition at different incremental distances (Δ) of images in the point (200', 200) and the focus analysis of said acquired images, the images having been taken with a gain value of the camera and aperture time such that said images all have a predetermined value of light intensity constant in the points (200', 200) and wherein, furthermore, the first focus distance (Z-foc-BI) at low magnification is used as starting level at which to carry out the acquisitions of images at different incremental distances for the determination of the punctual focus distance (Z-foc-punt) at a point, of the path (200', 200); - Determination of a second focus distance (Z-foc-AI) of the pit to said second magnification (A.I.) as a function of the punctual focus distances (Z-foc-punt) calculated at each single point of the path. - Selection of a lens at a first magnification (B.I.) and determination of a first focus distance (Z-foc-BI) at said first magnification (B.I.) for at least one pit (10, 10') of the slide (1, 1'), said focusing operation comprising the acquisition of images at different incremental distances (Δ) at a point (Po) predetermined of the matrix of the slide located outside of the pit and the focus analysis of said acquired images; - Subdivision of the pit under examination in a plurality of points (200', 200) defining an acquisition path of images; - Selection of a lens at a second magnification (A.I.) greater than the first magnification (B.I.); - For each point (200', 200) determination of a punctual focus distance (Z-foc-punt) at said second magnification (A.I.) through the acquisition at different incremental distances (Δ) of images in the point (200', 200) and the focus analysis of said acquired images, the images having been taken with a gain value of the camera and aperture time such that said images all have a predetermined value of light intensity constant in the points (200', 200) and wherein, furthermore, the first focus distance (Z-foc-BI) at low magnification is used as starting level at which to carry out the acquisitions of images at different incremental distances for the determination of the punctual focus distance (Z-foc-punt) at a point, of the path (200', 200); - Determination of a second focus distance (Z-foc-AI) of the pit to said second magnification (A.I.) as a function of the punctual focus distances (Z-foc-punt) calculated at each single point of the path.