© 2017 Elsevier Inc. Energy expenditure and metabolism, is a well-studied field as it is linked to many diseases as dysregulation of metabolism is associated with cancer, neurodegeneration, and aging. Classical methods of studying metabolism in vivo are well established, but most are tedious and expensive, thus, finding methods of accurately measuring metabolism in living organisms that is quick and non-invasive is of strong interest. In this work, we validate the use of resazurin; a compound that is conformationally changed into fluorescent resorufin upon metabolic reduction by NADH2, as a metabolic assay for adult zebrafish. This assay is based on the principle that increases in resorufin fluorescence intensity (FI) conveys relative changes in metabolic output of the organisms. We demonstrate the effectiveness of resazurin in measuring metabolic changes in zebrafish larvae and adults in relation to number of pooled fish, as well as temperature alteration. Moreover, we provide details on the appropriate and optimized diluents and concentrations of resazurin. Further, by using a novel sample collection technique, we can increase the temporal possibilities that were previously limited, as well as show that samples can be stored and measured at a later time point with no decrease in accuracy. Thus, the validation of this assay in adult zebrafish may increase the versatility and complexity of the types of experiments that can be performed and have many practical applications in the field.