Little is known about the levels of stress experienced by African buffalos affected by injury, disease, or socio-ecological and anthropogenic factors. To be able to start filling this gap, we examined the suitability of two 11-oxoaetiocholanolone enzyme-immunoassays (EIAs) detecting 11,17 dioxoandrostanes (11,17-DOA) as well as faecal glucocorticoid metabolites (FGMs) with a 5β-3α-ol-11-one structure (3α,11oxo-CM), respectively, for monitoring stress-related physiological responses in African buffalo. An adrenocorticotrophic hormone (ACTH) challenge in one male and one female housed at Mokopane Biodiversity Conservation Centre, South Africa, showed a threefold increase in circulating cortisol levels in a sample taken 40 min post-injection. Corresponding 11,17-DOA levels increased tenfold (female) and 15-fold (male) above baseline, and 3α,11oxo-CM concentrations increased ninefold (female) and 12-fold (male) above pre-injection levels, indicating that both EIAs are suitable for measuring FGMs in African buffalo.In addition, 11,17-DOA levels monitored during the adaptation process of individual housing revealed an up to 14-fold elevation in FGMs. Storage of faeces at ambient temperature for up to 16 h post-defecation resulted in an significant increase in 11,17-DOA levels 2 h after defecation. Finally, higher individual baseline 11,17-DOA concentrations were found in samples defecated overnight, indicating a possible diurnal effect in excretion of FGMs in African buffalo.