Stress in animals has been linked to behavioural and physiological changes in response to environmental, social and anthropogenic stimuli. Hence, stress-related responses in animals, especially in rodents, have been used as biological indicators of ecosystem health. This study aimed to establish an enzyme immunoassay (EIA) for monitoring adrenocortical activity in free-ranging Namaqua rock mice Micaelamys namaquensis (Rodentia: Muridae) using faeces as a prerequisite for assessing the effects of anthropogenic land use and season on faecal glucocorticoid metabolite (fGCM) concentration. Rodents were live-trapped seasonally across four land use types: an agricultural crop farm, an agricultural livestock farm, a human-populated site and a nature reserve; all situated in Limpopo Province, South Africa. Determined fGCM concentrations from capture and recapture events were used for biologically validating an EIA detecting steroids with a 5α-3β-11β-diol structure. Recapturing resulted in a significant overall 40% elevation of individual fGCM concentrations demonstrating the effectiveness of the chosen EIA to reliably detect glucocorticoid output in the study species. Neither land use type nor season affected fGCM concentrations in the species, suggesting that land use and season-related environmental changes do not necessarily act as stressors for M. namaquensis, presumably due to their adaptive and resilient nature. Such species can be used to identify ecosystems affected by human-mediated disturbances and allow insights into the management and restoration of these threatened ecosystems and their associated species.