Faecal hormone monitoring offers a robust tool to non-invasively determine the physiological stress experienced by an individual when faced with natural or human-driven stressors. Although already quantified for several species, the method needs to be validated for each new species to ensure reliable quantification of the respective glucocorticoids. Here we investigated whether measurement of faecal glucocorticoid metabolite (fGCM) provides a feasible and non-invasive way to assess the physiological state of sugar gliders (Petaurus breviceps), an arboreal marsupial native to Australia, by using both a biological and physiological validation. Our analysis confirmed that the cortisol enzyme immunoassay (EIA) was the most appropriate assay for monitoring fGCM concentrations in sugar gliders. Comparing the fGCM response to the physiological and the biological validation, we found that while the administration of ACTH led to a significant increase in fGCM concentration in all individuals, only six of eight individuals showed a considerable fGCM response following the biological validation. Our study identified the most appropriate immunoassay for monitoring fGCM concentrations as an indicator of physiological stress in sugar gliders, but also supports recent suggestions that, if possible, both biological and physiological stressors should be used when testing the suitability of an EIA for a species.