Androgens may play a key role in shaping the unique reproductive traits of male spinifex hopping mice (Notomys alexis), but little is known about the reproductive endocrinology of this species. Measurement of faecal androgen metabolites (FAMs) offers a non-invasive tool for monitoring testicular activity. Before applying this tool, physiological validation is required to demonstrate that changes in FAMs reflect changes in circulating testosterone for a given species. The goal of this study was to validate an enzyme immunoassay for monitoring FAMs in Notomys alexis. We compared the performance of two different assays (UVM-T and R156/7) for monitoring changes in FAMs following the administration of human chorionic gonadotropin (hCG) to stimulate androgen production by the testes. Both assays detected a significant increase in FAMs and had similar patterns. The UVM-T assay was more sensitive to changes in testicular activity, with a shorter excretion lag time and greater response magnitude. These findings indicate that we can reliably monitor testicular activity using faecal hormone metabolite analysis in Notomys alexis and can now utilise FAM measurements to better understand the species’ unusual reproductive biology.

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