The study of fish stress is usually assessed by measuring blood cortisol. Nevertheless, blood provides only a snapshot of the hormonal profile at one point in time. An alternative source of cortisol may be found in scales, providing a new approach for assessing long-term hormonal levels. The present study aimed to develop and validate a methodology for detecting cortisol in scales of goldfish (Carassius auratus). The study highlights the importance of an initial isopropanol washing procedure to completely eliminate external contaminations of cortisol. Additionally, the biochemical validation of the enzyme immunoassay verifies the ability to detect cortisol with repeatability and reliability in goldfish scales. In conclusion, this study provides validated information about a new methodology to measure cortisol in scales. The incorporation of this biomarker could provide retrospective hormonal measurements from species and time periods that are usually difficult or impossible to obtain, thus offering key data of an animal's physiology