The collection of non-invasive biological samples for the purpose of evaluating adrenal or reproductive hormones from free-ranging Australian mammals has been limited to date. This may be due to challenges associated with sample collection and validation as well as limited knowledge of these techniques. Metabolism, route of excretion and structure of the eliminated hormone metabolites can vary significantly between species. Therefore, regardless of sample type or hormone, a research program must start with a robust, biological validation process to ensure the sample collection, storage, processing, analysis and data evaluation methodologies are sound. Factors that will be reviewed include suitability of sample type, effects of environment exposure and sample storage on hormone stability, designing biological validation tests (e.g. using ACTH challenge or pregnancy to confirm biological relevance), determining excretion time delays, sampling intervals and sample numbers as well as testing different methods of sample processing, analysis and immunoassay antibody specificity to find the best fit for your species and research study. The potential application of these techniques are endless but, for example, a single faecal sample has been used to confirm pregnancy in species such as the ghost bat and dugong, allowing conservation biologists to better evaluate population fecundity towards improving population monitoring and management. With a good research plan, non-invasive hormone analysis can be a powerful tool for conservation biologists to gain information on reproductive status, adrenal health, and gender of individual animals to evaluate the overall health and fecundity of a wild population to improve conservation management and decisions.