Immunohistochemical analysis is a routine procedure for clinical and research studies in male fertility. However, most of the interpretations remain subjective and time-consuming, with inherent intra- and inter-observer variability. Given the prognostic and research implications of testicular assessment, a more objective and less time-consuming method is required. In the current study, we used in vitro matured pre-pubertal murine testes as a model. The main objective was to develop an affordable automated digital immunohistochemistry image analysis tool for an unbiased and quantitative assessment of testicular tissue sections. Testicular explants were fixed, cut, and stained for specific germ cell markers. The classical manual counting procedure was evaluated. Background and noise were reduced on brightfield images. Photomicrographs were stitched (Background_Elimination_Stitching) to create high-quality images. Two procedures were evaluated (IHC_Tool and Stained_Nuclear_Area); then a procedure (Necrotic_Area_Elimination) allowing withdrawal of the necrotic area observed after culture was assessed. Finally, the number of stained nuclei in the unaltered tissue area was extracted. The automated IHC_Tool procedure with images saved as TIFF at a ×200 magnification allowed the most rigorous cell quantification. IHC_Tool developed for testicular sample analysis can be used for various types of tissues. We foresee that this method will minimize inter-observer variations across laboratories and will be helpful for clinical trials and translational initiatives.Copyright © 2021 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.