Simple SummaryAnimal welfare is a main concern for captive animal keepers. The aim of this study was to evaluate the non-invasive vocalizations and sloughed skin scrape cortisol concentrations that are associated with potential stressful contexts in order identify the sources of stress in captive beluga whales. This study validated the use of beluga sloughed skin scrape as a matrix for measuring cortisol, measured the cortisol concentration using routinely collected samples, and identified the potential events (stressors) by examining medical and husbandry records. Besides, we estimated the time lag between the dates of the event and the peak in the cortisol concentration (response) in the skin samples, and analyzed the changes in the acoustic activities of the beluga whales during the selected events. The findings of this study may offer constructive recommendations regarding the environmental or internal stressors that captive cetaceans experience, and contribute to developing strategies to reduce or eliminate the stressors.AbstractAs people’s focus broadens from animals on farms to zoos and aquaria, the field of welfare science and the public’s concern for animal welfare continue to grow. In captive animals, stress and its causes are topics of interest in welfare issues, and the identification of an objective method that can be used to assess animals’ stress as a physiological state is essential. Both behavioral and physiological parameters can be used as indicators in order to assess animal stress quantitatively. To validate this approach, acoustic activity and the sloughed scrape skin cortisol concentration were used to evaluate the animal welfare of captive beluga whales (Delphinapterus leucas). The acoustic activity (5 min at 10:00 am) of three captiveD. leucaswas routinely recorded by a transducer and analyzed using audio editing software. The calls were separated into three main categories: whistles, pulses, and combo calls. The sloughed scrape skin samples were collected non-invasively once a week from all three animals’ fluke and/or flipper. Cortisol was extracted using a modified skin steroid extraction technique, and detected via commercially available enzyme immunoassays. The results showed that the cortisol concentration increased by varying levels when the whales encountered the same event. In addition, the number and distribution of the calls changed along with the events. This indicated that the changes in the cortisol concentration and acoustic behavior may have reflected the fluctuations in the environment and body condition. Therefore, the scrape cortisol measurement and acoustic recordings could be used to monitor stress levels in captive beluga whales. We recommend that aquaria consider incorporating skin scrape cortisol and acoustic activity monitoring into their standards for animal welfare.