A analytical method demonstrated the quantification of the hormone cortisol in cetacean skin. However, little is known about the transfer of hormones between blood and skin. Recognizing that such information is essential to effectively using skin samples within marine mammal stress research, the primary goals of this study were to (i) expand on the number of steroid hormones proved quantifiable in the cetacean skin matrix and (ii) validate the use of cetacean skin as a matrix for measuring stress-related hormones. Five adult bottlenose dolphins were subjected to an out of water stress test. Non-invasive sloughed skin samples were collected from each dolphin: once similar to 3 and once similar to 1 week prior to the stress test; at the time of the stress test; and twice weekly for 11 to 17 weeks subsequent to the stress test. LCMS/MS analysis of the samples recovered consistent data on three corticosteroids (cortisol, aldosterone, corticosterone), two androgens (testosterone, DHEA) and one progestagen (progesterone). A range of other hormones were also quantifiable, although not consistently so across samples. Results demonstrated that the hormonal response to an acute stressor could be detected in skin: the time from stress test to skin cortisol peak was an average of 46 days, whereas it was 55 days for corticosterone and 47 days for aldosterone. Results also showed that baseline hormonal concentrations were obtainable from skin samples collected during or immediately after the animals were subjected to the acute stressor. This study further develops and validates a non-invasive method for measuring cortisol and other hormones related to stress, health, and reproduction in the skin of cetaceans, potentially supporting investigations of acute and chronic stress, such as cetacean endocrine responses to distinct (e.g. naval sonar exposure) or prolonged stressors (e.g. shipping noise).