Objectives: The goal of this study was to use a validated assay to measure hair cortisol levels in healthy persons. Methods: For quantitative detection of hair-cortisol, a high-performance liquid chromatography-tandem mass spectrometry approach was established and validated. Hair samples were incubated in methanol for 16 hr at 52°C, after which the clear supernatant was sonicated and transfer to a clean tube, where it was dried. The residual sample dissolved in 100 μl methanol containing cortisone (internal standard) and injected. The average extraction recovery was calculated (90.8%). A mobile phase of two mM ammonium acetate (pH 4.2) and acetonitrile (50:50, v:v) was supplied at a flow rate of 0.3 ml/min on an Atlantis dC18 column (2.1 x 100 mm, 3 μm particle size). Multiple reaction monitoring was used in electrospray positive ion mode for mass spectrometry acquisition (m/z: 363.1 → 121.0 and 361.8 → 163.11 for cortisol and cortisone, respectively). Results: Cortisol and internal standard exhibited retention times of about 1.34 and 1.39 min, respectively. In the range of 2-100 ng/ ml, the relationship between cortisol level and peak area ratio of cortisol to cortisone was linear, with a detection limit of 0.3 ng/ml. The inter-day coefficient of variation and bias were 7.8% and 11.9%, respectively. 95% of the 88 men had cortisol detectable in their hair samples [mean (SD) length =1.3 (0.5) cm, weight = 33.9 (9.9) mg] and measurable in 49%, with a mean (median) 12.4 (8.7) pg/mg of hair. The method utilized for determining the stability and assessing the level of hair cortisol in samples taken from healthy adult men. Conclusion: For hair cortisol measurements in clinical labs, the described test is reliable, exact, and relevant.