Although bats are the second most species-rich mammalian order, very little is known about their endocrine physiology. Glucocorticoids (GCs) are commonly associated with the stress response, but also modulate vital physiological functions which help animals adapt to their environment. Understanding normal patterns of adrenocortical activity can provide valuable insights into a species’ fitness. Non-invasive hormone monitoring via faecal samples provides an integrated measure of adrenocortical activity while minimising stress on the animal but must be properly validated to ensure reliable results. The goal of this study was to validate an enzyme immunoassay for monitoring faecal glucocorticoid metabolites (FGMs) in a common Australian insectivorous bat species, the Gould’s wattled bat (Chalinolobus gouldii). We compared the performance of five assays for monitoring changes in FGMs following capture and transfer of C.gouldii from the wild to captivity. Four of the five assays detected a significant increase in FGMs following capture, but the magnitude of the increase and consistency across individuals differed considerably. We selected the UVM-69a assay as the best performing assay to then describe normative patterns of adrenocortical activity in the species. Males had higher FGM levels than females, and juveniles had higher FGM levels than adults. Individuals with poorer body condition had higher FGM levels. We also demonstrate seasonal patterns of FGMs with higher levels in March and April corresponding with reproductive up-regulation and lower levels in May and November. Our study is the first of its kind to examine adrenocortical activity in an Australian insectivorous bat and provides a valuable tool for studying this species. Understanding adrenal function in common species such as C.gouldii can shed light on the physiological mechanisms facilitating survival and success in changing environments.