Context In the past decade, flow cytometry has become a useful tool for evaluating cellular viability characteristics for non-domestic animals such as non-human primates, marine animals, and birds. This technology has the potential to vastly improve sperm-quality assessments, concentration counts and cell sorting in a more time-efficient and reliable manner. Aims The study aimed to validate the efficacy of using flow cytometry for amphibian sperm by comparing its results with those obtained through traditional means of sperm-quality assessment. Methods Sperm samples were collected from testes macerates of the African clawed frog (Xenopus laevis ) and subjected to both flow cytometry and microscopy analyses. Flow cytometry allowed for the simultaneous assessment of sperm viability and concentration by using fluorescent probes, whereas microscopy provided a traditional means of assessing sperm characteristics. Key results Sperm concentrations measured by flow cytometry and fluorescent microscopy were highly correlated, although flow cytometry methods estimated higher concentrations. Sperm viability measured by flow cytometry and that measured by fluorescent microscopy were not significantly correlated and were significantly different, varying by only ~8% in viability, on average. Conclusions Although flow cytometry overestimated concentration and live/dead assessments, the discrepancies were slight enough to indicate that flow cytometry can still be a valuable method for assessing amphibian sperm. Implications These results validated the utility of flow cytometry as a reliable tool for assessing amphibian sperm viability and concentration, offering a promising alternative to traditional, time-consuming methods.