Monitoring wildlife stress levels is essential to ensure their quality of life in captivity or in the wild. One promising method to assess the stress response is the comeasurement of glucocorticoids (GC) and dehydroepiandrosterone sulfate (DHEAS), adrenal hormones involved in the modulation of the stress response. Although noninvasive methods to measure GCs have been validated in several species, only a few studies have validated DHEAS assays. The aims of this study were (1) to describe an enzyme immunoassay (EIA) to measure DHEAS levels, (2) to validate this assay for fecal samples in gibbons and siamangs, and (3) to test hormonal stability after one freeze-thaw cycle and over time at two freezer temperatures (−20°C and −80°C). Subjects included 32 gibbons and siamangs from U.S. zoological parks. The EIA was validated analytically by parallelism and accuracy tests, and biologically by confirming a DHEAS response 1-2 days after a stressful event (accident, vaccination, or transportation) in three individuals. In addition, fecal DHEAS levels in a pregnant female were above nonpregnant/nonlactating levels and declined progressively the following parturition. The hormonal stability experiments revealed no significant changes in fecal DHEAS levels after one freeze-thaw cycle.Hormonal levels in fecal extracts were stable for 2 months, regardless of the storage temperature, with no significant differences between −20°C and −80°C conditions. The EIA described has high sensitivity and it is suitable for fecal DHEAS measurement in gibbons and siamangs, with a potential to be applied to other species.