The presence of Ethyl glucuronide (EtG) in hair provides a strong indication of ethanol consumption and its investigation is of interest in both clinical and forensic contexts because of the wide window of detection. However, due to the possibility of false negative results in cases of small ethanol intake or excessive hair washing, the combined measurement of ethyl palmitate (EtP) with EtG could be useful. In this study, a sensitive UHPLC-MS/MS procedure for the measurement of EtG in hair was developed and validated, using optimized sample preparation and chromatographic separation. Milled hair was extracted with water for 24 h at room temperature, followed by clean-up of the extract by ion-exchange solid phase extraction (SPE). Extraction was highly efficient, with yield of 96.93-101.06%. Chromatographic separation was performed with a Fluoro-Phenyl stationary phase. The assay was linear from 4 to 500pgmg, with accuracy in the range of 100.30-106.16%. Matrix effects (-0.87 to 5.89%) were adequately compensated by the use of deuterated EtG as internal standard. EtG was measured in hair samples of 46 volunteers, and results were compared with hair concentrations of ethyl palmitate (EtP) and the score in the AUDITC questionnaire. EtG hair concentrations were significantly correlated to the AUDIT-C classification (r=0.365, p<0.05), but not to EtP hair levels. The diagnostic performance of EtG hair concentrations to identify excessive or moderate ethanol use was similar to the capability of AUDIT-C to identify severe and high health risk (Kappa, p=0.013). The developed assay is suitable for clinical use, providing a useful tool to evaluate chronic ethanol consumption.