Although efforts are being made to stop earth’s sixth mass extinction, 1 out of 4 species is still at risk. To improve the in situ and ex situ conservation strategies of endangered species, a better understanding of their reproductive biology and adaptability is crucial. The latter is best achieved via non-invasive hormone monitoring, with immunoassays (EIA) being the most accessible tool. However, their use is complicated by the species’ specificity of hormone metabolism. In this study we aim to install a wildlife monitoring platform by: (1) developing a steroidomics UHPLC-HRMS methodology for screening of hormones and their metabolites in urine/fecal samples (WP1), (2) establishing a workflow for the identification of biomarkers or fingerprints to diagnose/predict fertility, pregnancy and adaptivity and consequently develop fast and hands-on monitoring methods (WP2+3), (3) offering a true gold standard reference method for the validation of existing and new immunoassays (WP4). As a proof-of-concept, the platform will be validated based on 4 pilot studies. These studies include a variety of species, i.e. Asiatic golden cat, Iberian lynx, giant panda and white rhinoceros, and are focused on research questions related to both reproductive and adaptive biology of wildlife species. The foundation of this high-tech wildlife monitoring platform, unique in Europe, will strengthen basic and fundamental wildlife conservation research.