The ability to non-invasively monitor stress hormone levels in free-ranging animals could significantly aid in conservation and management efforts. Our objective in this study was to demonstrate the effectiveness of assay of fecal corticoid metabolites in detecting a stress response in cougars (Puma concolor). Fecal samples were collected from 9 captive cougars before and after an artificial stressor. Steroid hormones were extracted from the samples. Adrenal corticoid metabolite concentrations of the resulting extracts were quantified using cortisol and corticosterone assays. Results indicated that fecal glucocorticoid metabolite levels increased significantly 24 hours after the stressor in 6 of the 9 individuals. Behavior correlated with the hormonal response; all cougars that displayed a flight response to the stressor also had elevated stress hormone measures. Elevated levels of stress hormones were not observed in individuals that did not attempt to flee. We have demonstrated with this study that measurement of fecal hormone metabolites is sufficiently sensitive to detect an adrenal response to stress in cougars and could be applied in the field to monitor stress levels in free-ranging populations.