<p>Cortisol is the main corticosteroid hormone involved in the regulation of stress status in teleost fish and a recognized biomarker of stress, whose plasma concentration increases significantly in response to a perceived hazard situation. Traditional methods to quantify cortisol levels are usually performed through blood extraction, but this approach may be considered as a highly severe stress-inducing method for fish, damaging tissues and inflicting even more stress; therefore, interest in developing alternative and less severe techniques has arisen. In this sense, this study aimed to validate that methods to quantify cortisol in skin mucus and fin of mullet juveniles held in captivity after exposure to an acute stress were as efficient as in plasma samples to confirm physiologically fish behavioural characterization into proactive and reactive stress coping styles (SCS). Flathead grey mullet <i>Mugil cephalus</i> juveniles were characterized as proactive and reactive SCS through group and individual behavioural tests, and cortisol levels were quantified in plasma, skin mucus and fin before (control) and after (in proactive and reactive fish) exposure to an acute stress. Results demonstrated that cortisol quantification in skin mucus and fin was as accurate, less severe, reliable and efficient as in plasma to characterize proactive and reactive SCS in this fish species. Moreover, these techniques might contribute to welfare of individuals.</p>