Measuring physiological stress reactions through the quantification of plasma cortisol often involves physical restraint, which acts as a stressor itself. Here, we present the validation of a non-invasive method for assessing adrenocortical activity as an indicator of stress in the bat-eared fox (Otocyon megalotis). By conducting an adrenocorticotropic hormone (ACTH) challenge, we examined the suitability of three enzyme immunoassays (EIAs) detecting 11,17 dioxoandrostanes (11,17-DOA) as well as faecal glucocorticoid metabolites (fGCM) with a 5β-3α-ol-11-one (3α,11oxo-CM), or 11,17,21-trihydroxy-4-ene-20-one structure (cortisol), respectively, for monitoring stress-related physiological responses in male and female bat-eared foxes. Our results suggest that the cortisol EIA seems most suitable for measuring fGCMs in this myrmecophageous mammal. Using the cortisol EIA, we compared fGCM concentrations of three populations of foxes. Only one population experienced a sudden change in social environment and the assay appeared to effectively detect the expected resulting increase in stress hormone levels. Therefore, the identified EIA is effective at detecting intraspecific variation in fGCM levels and hence is a useful tool to evaluate physiological stress responses in this species.