To develop non-invasive techniques for monitoring steroid stress hormones in the feces of free-living animals, extensive knowledge of their metabolism and excretion is essential. Here, we conducted four studies to validate the use of an enzyme immunoassay for monitoring fecal cortisol metabolites in snowshoe hares (Lepus americanus). First, we injected 11 hares with radioactive cortisol and collected all voided urine and feces for 4 days. Radioactive metabolites were recovered predominantly in the urine (59%), with only 8% recovered in the feces. Peak radioactivity was detected an average of 3.5 and 5.7 h after injection in the urine and feces, respectively. Second, we investigated diurnal rhythms in fecal cortisol metabolites by measuring recovered radioactivity 2 days after the radioactive cortisol injection. The total amount of radioactivity recovered showed a strong diurnal rhythm, but the amount of radioactivity excreted per gram of feces did not, remaining constant. Third, we injected hares with dexamethasone to suppress fecal cortisol metabolites and 2 days later with adrenocorticotropic hormone to increase fecal cortisol metabolites. Dexamethasone decreased fecal cortisol metabolites concentrations by 61% and adrenocorticotropic hormone increased them by 1,000%, 8-12 h after injection. Fourth, we exposed hares to a simulated predator (dog). This increased the fecal cortisol metabolites concentrations by 175% compared with baseline concentrations 8-12 h after exposure. Thus, this enzyme immunoassay provides a robust foundation for non-invasive Weld studies of stress in hares.