Hair cortisol has been proposed as the best biomarker in order to evaluate the Hypothalamic-Pituitary-Adrenal (HPA) axis. It gives information about the hormone levels to which the individual was exposed in the previous three months. The current available methodologies are mass spectrometry, which is expensive and inaccessible in the daily laboratory, and ELISA which is a non-automatic method with low precision. The aim of the present study was to validate an automated hair cortisol measurement method to be used in daily laboratory, its comparison with the reference method and its application in diverse clinical situations. Methods: Hair cortisol levels were measured with Siemens Immulite 2000 automated CLIA analyzer modified after extracting samples with methanol. Limit of blank (LOB), limit of detection (LOD) and limit of quantification (LOQ) were performed according to the EP-17A protocol. The precision profile was determined using four hair extracted samples of different cortisol concentrations (1.6 nmol/L, 3.9 nmol/L, 11.5 nmol/L, 20.0 nmol/L). Ten samples were measured by the proposed method and compared with mass spectrometry. Hair cortisol was measured in a total of 222 healthy individuals; normal reference values were obtained from 203 healthy individuals without medication, adrenal pathology and free of stress according to Holmes-Rahe life events scale score. The remaining 19 individuals presented Holmes-Rahe life events scale score >300 (stressed individuals). The new method was used to evaluate hair cortisol levels in different pathologic situations such as acute myocardial infarction (AMI, n=17), Cushing syndrome (CS, n=7) and poliquistic ovarian syndrome (PCO, n=20). Results: Regarding the validation of the method, LOB was 0.9 nmol/L; LOD was 2 nmol/L and LOQ was 3.4 nmol/L. Precision profile variation coefficients were 45.7% for 1.6 nmol/L, 16.5% for 3.9 nmol/L, 8.3% for 11.5 nmol/L, and 6.7% for 20.0 nmol/L. The presence of cortisol in methanol extracts was verified by tandem mass spectrometry. In healthy free stress individuals median was 54 pg/mg hair (range 40-173) and 250 pg/mg hair (range 182-520 pg/mg) in stressed individuals. Reference cut off value was 173 pg/mg hair. Hair cortisol in Cushing patients was 334 pg/mg hair (range 197-3588 pg/mg). Remarkably 41% (7/17) of AMI patients presented hair cortisol over the reference value (323 pg/mg ( range 182-903)) as well as 45% (9/20) of PCO patients (327 pg/mg hair (range 176- 569)), both significantly higher than healthy individuals (9.5%, Chi-Square test p<0.001). Conclusions: A novel technique which allows automated hair cortisol measurement was developed. The results support the possibility to use this method in clinical laboratories for evaluating and monitoring chronic stress and clinical application in different pathologies. This procedure has been patented by the University of Buenos Aires.