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Abstract Minimally invasive methods for estimating hormone concentrations in wild vertebrates offer the opportunity to repeatedly measure behavior and hormone concentrations within individuals while minimizing experimenter interference during sample collection. We examined three steroid hormones (corticosterone, CORT; 17-b estradiol, E2; progesterone, PROG) in túngara frogs (Physalaemus pustulosus) using non-invasive water-borne methods. Using solid-phase extraction of water samples and liquid extraction of plasma and homogenate samples, coupled with enzyme immunoassays, we complimented the conventional validation approaches (parallelism, recovery determination) with dose-response assays that incorporated pharmacological challenges with adrenocorticotropic hormone (ACTH) and human chorionic gonadotropin (HCG). We also compared steroid concentrations in water to those observed in plasma and whole body homogenates. Lastly, we identified the constituent steroids in each sample type with a panel targeting 30 steroid species using high performance liquid chromatography-mass spectrometry (HPLC-MS). We found that a 60-min water-bath captures physiologically relevant changes in concentrations of CORT, E2 and PROG. Peak levels of water-borne CORT were found at approximately 2 h after ACTH injection. Water-borne CORT and E2 concentrations were positively correlated with their plasma and homogenate equivalents, while water-borne PROG was uncorrelated with homogenate PROG concentrations but negatively correlated with homogenate E2 concentrations. Together, our findings indicate that sampling water-borne hormones presents a non-invasive and biologically informative approach that will be useful for behavioral endocrinologists and conservation physiologists.