Quantifying physiological stress may aid in a better understanding of how animals survive various environmental conditions. One noninvasive technique for assessing physiological stress in animals is to extract steroid hormones from fecal samples. This technique has the potential to aid in wildlife conservation by providing a better understanding of behavior and welfare for a variety of species. The objectives of the study were to (1) determine responses in plasma and fecal steroid hormone concentrations for coyotes (Canis latrans), (2) determine which steroid hormone (cortisol or corticosterone) was better for examining physiological stress responses for coyotes, and (3) determine the amount of time steroid hormone metabolites can be found in coyote scats during 2 different seasons. We first conducted an adrenocorticotropic hormone (ACTH) challenge in 16 coyotes and examined both plasma and fecal steroid hormone concentrations. An ACTH challenge is when there is an externally derived hormone (ACTH) injected into a subjects blood stream, which causes an increase in the subject’s circulating steroid hormones associated with physiological stress. We injected 16 treatment animals with ACTH and 16 control animals with a saline solution. We collected blood and fecal samples pre- and post-injection to measure steroid hormone concentrations. Radioimmunoassay, a laboratory method used to measure substances, was used to measure concentrations of steroid hormones in coyote blood and feces. To evaluate if steroid hormone concentrations remained in feces for an extended period of time we collected samples from 6 captive coyotes and left the samples in natural environmental conditions for 13 days. Each day a sub-sample was collected, and hormones were extracted and run through radioimmunoassay. We found increased steroid hormone concentrations after an ACTH challenge in both blood and feces, validating the use of fecal steroid hormone concentrations as a tool to measure physiological stress in coyotes. We also found there were no differences in levels of steroid hormone concentrations over 13 days. Our study provides validation for use of fecal steroid hormone concentrations in coyotes to quantify stress levels and confirms that steroid hormone metabolites are viable up to 13 days post deposition in coyote scat. This noninvasive tool can aid in the evaluation of the abilities of coyotes to adapt and exist in a variety of habitats.