AbstracteDNA-based methods represent non-invasive and cost-effective approaches for species monitoring and their application as a conservation tool has rapidly increased within the last decade. Currently, they are primarily used to determine the presence/absence of invasive, endangered or commercially important species, but they also hold potential to contribute to an improved understanding of the ecological interactions that drive species distribution. However, this next step of eDNA-based applications requires a thorough method development.We developed an eDNA assay for the white-clawed crayfish (Austropotamobius pallipes), a flagship species of conservation in the UK. Multiple subsequent in-situ and ex-situ validation tests aimed at improving method performance allowed us to apply eDNA-based surveys to evaluate interactions between white-clawed crayfish, crayfish plague and invasive signal crayfish.The assay performed well in terms of specificity (no detection of non-target DNA) and sensitivity, which was higher than more established traditional species survey methods. Quantification of species biomass was, however, less reliable.Comparison of eDNA sampling methods (precipitation vs. various filtration approaches) revealed that optimal sampling method differed across environments and might depend on inhibitor concentrations.Finally, we applied our methodology together with established assays for crayfish plague and the invasive signal crayfish and demonstrated their significant interactions in a UK river system.Our analysis highlights the importance of thorough methodological development of eDNA-based assays. Only a critical evaluation of methodological strengths and weaknesses will allow us to capitalise on the full potential of eDNA-based methods and use them as decision support tools in environmental monitoring and conservation practices.