Understanding the migratory cycle of the European eel is crucial for implementing effective conservation measures. The reasons why some glass eels settle in lower estuaries rather than migrating upriver remain unclear. This study aims to identify metabolomic signatures that distinguish active (migrant) from inactive (nonmigrant) glass eels. Using a combination of target and non-target screening (NTS) approaches, the metabolite profile of glass eels was studied, and a PLS-DA classification model was applied to find differences between behavioural phenotypes. This model highlighted methionine, glutaryl-L-carnitine, and palmitoylcarnitine as key metabolites, with methionine being significantly different between groups. Glutaryl-L-carnitine strongly correlated with activity, suggesting it might be a more sensitive indicator of glass eel activity than previously studied parameters such as weight loss and oxygen consumption. The findings suggest that differences between active and inactive eels result from both swimming activity and intrinsic metabolic differences, with methionine linked to both factors. We also explored potential differences in how diazepam affects active and inactive glass eels. However, our metabolomic approach lacked the sensitivity to detect significant variations. Overall, this study provides valuable insights into the metabolomic distinctions between active and inactive glass eels, establishing a foundation for future research in this field.