<p>Hair cortisol concentration is a retrospective, long-term measure of cortisol secretion, often used as a biomarker for chronic stress or suspected cyclic Cushing’s syndrome. Various methods for hair cortisol extraction, typically involving intact, minced, or milled hair incubated with methanol or extraction buffers, may not achieve complete extraction. Incomplete extraction can bias results, particularly when comparing hair samples of differing thickness and texture. Additionally, sample-to-sample variation might be caused by inconsistent cutting or grinding of the hair by different technicians. We aimed to achieve complete cortisol extraction through the enzymatic digestion of hair. After digestion and sample clean-up, we analyzed the final extracts using two-dimensional liquid chromatography-mass spectrometry. Complete digestion was observed with an overnight enzymatic reaction. This method demonstrated high reproducibility, with an intra-day precision of 3.6%, and inter-day precision of 6.5% in a homogenized pool. Moreover, duplicate CV% of intact hairlocks ranged from 0.1% to 8.9%. Our protocol does not influence the cortisol concentration. It is less labor-intensive and more consistent than mincing or milling hair. Our extraction yield was higher compared to the commonly used methanol extraction method. Samples extracted with methanol showed, on average, 19% lower cortisol concentrations than those measured in digested aliquots. We have developed and validated a new, superior method for extracting cortisol from hair utilizing enzymatic digestion. This strategy provides a more thorough extraction, greater consistency, and requires less labor than traditional methods.</p> Graphical Abstract <p></p>