Spermatogenesis, the process of germ cell development into mature spermatozoa, occurs within the seminiferous tubules. It includes mitosis, meiosis, and postmeiotic differentiation phases involving intricate cellular transformations. The seminiferous tubules, comprising 12 longitudinally organized stages of the seminiferous cycle, provide a dynamic environment for germ cell development. At any given stage, the seminiferous epithelium consists of spermatogonia, spermatocytes, and spermatids at a particular point in differentiation. Identifying stages is critical for understanding the dynamic changes of a protein of interest or for evaluating at what point in differentiation a particular gene mutation impacted spermatogenesis. This protocol outlines a transillumination-assisted microdissection method enabling precise staging of seminiferous tubules freshly isolated from mice. This method takes advantage of the unique light absorption patterns along the length of the tubule, as well as the fact that the 12 stages occur in a sequence, allowing for dissecting out each stage. Additionally, the protocol includes immunostaining for the acrosomal marker SP-10 to confirm staging accuracy. While described in the context of mice here, the method is applicable to other animal models and will be particularly useful in evaluating spermatogenesis in mutant mice or those exposed to toxic chemicals.