When pathologists analyze the cytopathological assessment process, they firstly find the optimum focused region by moving the microscope table along the Z axis. Auto-focusing provides a better and more effective imaging by reducing the personality dependence on the microscope system. Various studies have been carried out in the literature to determine the optimum auto-focus function. The aim of the study is to determine the optimum function for cytopathological assessment processes outside the areas investigated in the literature. In order to find the optimum focus function in the study, 12 different focus functions were tested under 5 different evaluation criteria (execution time, accuracy, number of local maximum points, range, noise level). A total of 12 different image series were created for each of the test operations using different magnification objectives (10x - 20x - 40x). According to the results obtained, it is decided that the optimum auto-focusing function for the cytopathology assessment processes is Variance.