IntroductionValid quantification of fecal corticosterone metabolites (FCM) is a valuable non-invasive method for assessing hypothalamic-pituitary-adrenal (HPA) axis activity and stress responses in animals. The increasing emphasis on animal welfare highlights the need not only to expand knowledge but also to critically evaluate methodological approaches, particularly with regard to how individual procedural steps may influence results and contribute to variability or bias. Methodological inconsistencies between studies can significantly affect the accuracy and comparability of results. This study aimed to optimize the extraction procedure of corticosterone metabolites from feces of domestic rabbits (Oryctolagus cuniculus domesticus).MethodsWe experimentally compared the effects of sample weight (50, 100, and 500 mg), extraction protocol (one-step vs. two-step), and dilution factor (10 × and 100 × ) using 80% methanol and commercial ELISA kit.ResultsThe optimal performance, in terms of extraction efficiency and reproducibility, was achieved using 100 mg of lyophilized and homogenized feces in a two-step methanol extraction with 3 + 2 mL. This configuration yielded a recovery rate of 51.55% and minimized matrix interference. Lower or higher sample weights resulted in technical artifacts or low extraction efficiency.DiscussionThe optimized method provides a standardized, reproducible, and feasible approach for the robust non-invasive assessment of adrenocortical activity in rabbits and other small mammals. These results underscore the necessity of detailed methodological reporting to ensure data validity and comparability of results across studies, assuming the same immunoassay is used.