With the world currently facing a global amphibian extinction crisis, the development of techniques to help meet the needs of conservation managers and researchers studying the reproductive biology of amphibians is needed. Here, we developed enzyme immunoassays to measure estrone, testosterone, and progesterone hormone metabolites in the urine of Litoria raniformis, the southern bell frog. Concentrations of urinary estrone, testosterone, and progesterone increased during the breeding season for females (P<0.05). Concentrations of urinary testosterone and progesterone increased for males during the breeding season compared with that for months where no reproductive behaviors were observed (P<0.05). Furthermore, urinary estrone concentrations proved to be a reliable sexing tool for adult frogs, with no overlap between the sexes in 98% of cases, regardless of season. There was no difference in estrone (P=0.204) or testosterone (P=0.485) metabolite concentrations between samples taken immediately upon capture and those taken 12 to 24h later from the same individual. Progesterone metabolite concentrations were lower on Day 2 than upon collection (P=0.004). This is the first study to show that urinary hormone analysis can be a useful technique for reproductive monitoring in an amphibian. Additionally, hormone metabolite measures offer promise as sex identification tools for monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.