Leopards utilizing areas under different anthropogenic influences may be exposed to different environmental, physiological and psychosocial stressors. Given the elusive nature of the species, a non-invasive approach to monitor responses to stressors would be advantageous to date; however, no test system has been established to determine glucocorticoid concentrations in leopard faeces. The study aimed to examine the suitability of five different enzyme-immunoassays (EIA’s) for monitoring adrenocortical function in the leopard based on faecal glucocorticoid metabolite (fGCM) analysis. After performing an adrenocorticotropic hormone (ACTH) stimulation test, examining gastrointestinal transit (GIT) time under different feeding regimes and investigating the stability of fGCM concentrations post-defecation, faecal samples were collected from free-ranging leopards in a peri-urban and conservation area. An EIA using an antibody against 5α-pregnane-3β,11β,21-triol-20-CMO:BSA seems most suitable for assessing adrenocortical function in male and female leopards, with post-stimulation fGCM concentrations increasing by 331% (male) and 203% (female) respectively. GIT varied distinctly between the two different facilities tested, and fGCM concentrations post-defecation remain fairly stable for up to 6 days. Faecal GCM concentrations did not vary between the two study sites (T7,37=187, p=0.35). Overall median fGCM concentration of female leopards were ~50% and ~250% higher compared to males at both sites, respectively. Females also demonstrated higher variability in individual fGCM concentrations compared to males, which might be linked to differences in reproductive status. The established method now adds to the tools available to address some of the wildlife management, conservation and human-predator mitigation measures for free-ranging leopards under different land use practices.