We present the development of the first procedure for hair cortisol measurement through an automated method. Hair samples were obtained from 286 individuals. After cortisol extraction, samples were measured in a Siemens Immulite 2000 (Gwynedd, UK) automated chemoluminiscent immunoassay analyzer. Normal reference values were obtained from hair cortisol levels measured in 213 healthy individuals with low levels of stress. Hair cortisol concentration median was 55 pg/mg hair (2.5-97.5 percentile (40-128)) in healthy individuals with low levels of stress and 250 pg/mg hair (range 182-520) in stressed individuals. No significant differences were observed in hair cortisol levels between subjects with and without dye (40 (40-107) and 40 (40-155) pg/mg hair, respectively; p = 0.128). The novel procedure presented here shows an adequate analytical performance.