Fecal glucocorticoid metabolites (FGMs) are commonly used as indicators of an animal's stress response in behavioral and ecophysiological studies. FGM assays provide a noninvasive and efficient means of assessing adrenocortical activity. We used 12 African buffalo (Syncerus caffer) temporarily maintained in an enclosure to evaluate 2 commercially available FGM assays as tools for assessing stress levels in buffalo in field studies. We also used the experiment to assess potential adverse effects of immobilizations on the study animals. Buffalo responded rapidly to stimulation with adrenocorticotropic hormone (ACTH), which stimulates the adrenal cortex to secrete glucocorticoids. ACTH-stimulated buffalo had higher plasma cortisol concentrations than saline-injected controls between 5 min and 1 h after injection. The ACTH-induced plasma cortisol peak was detectable in FGMs at 10–20 h post-injection. Both of the commercial test kits we evaluated were capable of detecting the ACTH-induced peak in FGM. However, the radioimmunoassay delivered more consistent detection across weeks than the enzyme immunoassay. We tested whether immobilization and handling elicited a stress response detectable by FGM, by comparing immobilized, saline-injected buffalo with controls that were not immobilized or handled. Adult buffalo mounted a stress response to immobilization and handling, whereas subadults did not, suggesting an age-related difference in response to chemical immobilization. Our study validates use of commercially available kits for quantifying FGMs under field conditions.