Assessing oxidative stress levels in spermatozoa provides crucial information on the health of the cell, while also potentially providing insight into the systemic health of the individual. Indeed, elevated oxidative stress in spermatozoa has been implicated in compromising sperm function and, consequently, is now considered one of the leading mediators of numerous male reproductive pathologies. Here we describe two flow cytometric assays: Dihydroethidium and Mitosox Red, which can be used to detect reactive oxygen species originating from the cytosol and mitochondria, respectively, in both mouse and human spermatozoa. Such tools have utility in studies investigating the impact of reproductive aging, toxicants, and environmental exposures on the male germline. These assays are also beneficial in the context of testing novel pharmaceutical interventions or antioxidant defence therapies aimed at preserving fertility or for use in assisted reproduction technologies.