In South Africa, blue wildebeest (Connochaetes taurinus) are routinely captured for relocation purposes. To monitor the stress caused by this practise, a non-invasive method assessing adrenocortical function as a measure of stress would minimize disturbance during sample collection. In our study, an adrenocorticotropic hormone challenge, and a mass-capture event were used to examine the suitability of five enzyme immunoassays (EIAs) for monitoring stress-related physiological responses using faeces as a sample matrix. The tested 11-oxoaetiocholanolone I EIA performed best, showing a 2126% increase above baseline after 22 h for a male, and a 474% increase for a female 23 h post-ACTH injection. Baseline faecal glucocorticoid metabolite (fGCM) concentrations did not differ between animals captured on either day 1 or day 2 of the capture event, indicating that the frequent presence of a helicopter during the two days did not influence fGCM concentrations. However, during capture-related restraint for up to 10 h, an overall 1.5-fold elevation in (fGCM) concentrations was found. Storage of faeces at ambient temperature post-defecation indicated a fair stability of fGCMs for up to 8 h. The ability to reliably assess adrenocortical function provides a solid basis to examine endocrine responses to putative stressful circumstances in blue wildebeest.