Cortisol is the main glucocorticoid released during stress responses in most fish and has been employed to investigate different stressors, including organic pollutants. This review discusses shifts in cortisol concentrations and examines different matrix sampling methods (invasive vs. minimally or non-invasive) and the main analytical cortisol determination techniques (immunoassays and liquid chromatography-tandem mass spectrometry). Assessments on organic pollutant exposure in fish and associated adverse effects are also discussed. Studies in this regard may aid in identifying organic pollutant toxicological modes of action, mechanistic response, toxicokinetics, and toxicodynamics, as well as pollution sources and associated health risks in fish, ultimately aiding in the development of effective management strategies to mitigate the impacts of organic pollutants on fish populations and their associated ecosystems.