Water-borne hormone measurement is a noninvasive method suitable for amphibians of all sizes that are otherwise difficult to sample. For this method, containment-water is assayed for hormones released by the animal. Originally developed in fish, the method has expanded to amphibians, but requires additional species-specific validations. We wanted to determine physiological relevance of water-borne corticosterone in spotted salamanders (Ambystoma maculatum) by comparing concentrations to those taken using established corticosterone sampling methods, such as plasma. Using a mixture of field and laboratory studies, we compared water-borne corticosterone levels to other traditional methods of sampling corticosterone for spotted salamander larvae, metamorphs, and adults. Despite multiple attempts, and detecting differences between age groups, we found no correlations between water-borne and plasma corticosterone levels in any age group. Water-borne sampling measures a rate of release; whereas plasma is the concentration circulating in the blood. The unique units of measurement may inherently prevent correlations between the two. These two methods may also require different interpretations of the data and the physiological meaning. We also note caveats with the method, including how to account for differences in body size and life history stages. Collectively, our results illustrate the importance of careful validation of water-borne hormone levels in each species in order to understand its physiological significance.