A system and a method for measuring the focus state of an optical instrument (for example, a microscope) are described. The system and the method provide for forming an image of the exit pupil of theobjective of the optical instrument. Where the image of the exit pupil is formed, one or more optical elements (for example a prism, or mirrors) are placed, deviating at least part of the rays comingfrom the exit pupil, so that rays coming from different non-overlapping portions of the pupil follow separate optical paths. The rays coming from the two portions of the pupil are then focused, in order to obtain two bidimensional images. A computer determines the mutual distance between these two bidimensional images as a mutual rigid lateral displacement between the two and, based on this distance, determines the corresponding defocus of the optical instrument.