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Background: Hair analysis for chronic excessive alcohol (ethanol) use has focused on ethyl glucuronide (EtG), a minor metabolite of ethanol. Preferred methods have involved high-performance liquid chromatography (HPLC) combined with tandem mass spectrometry (MS/MS) in line with an electrospray ionization (ESI) source. EtG analysis in hair has not yet been introduced to Thailand, Objective: To validate an in-house HPLC-ESI-MS/MS hair analysis protocol for EtG and to apply it to a field sample of alcohol drinkers to assess different risk levels of alcohol consumption as measured by the Alcohol Use Disorders Identification Test (AUDIT)., Material and Method: Validation procedures followed guidelines of the US Food and Drug Administration, the European Medicines Agency, and the Scientific Working Group for Forensic Toxicology. One hundred twenty subjects reported consuming alcohol during a 3-month period prior to enrollment. After taking the Thai-language version of AUDIT, subjects were divided on the basis of test scores into low, medium, and high-risk groups for chronic excessive alcohol use., Results: The protocol satisfied the international standards for selectivity, specificity, accuracy, precision, and calibration curve. There was no significant matrix effect. Limits of detection and quantification (LOD/LOQ) were set at 15 pg of EtG per mg of hair. The protocol was not able to detect EtG in low-risk subjects (n = 38). Detection rates for medium-risk (n = 42) and high-risk subjects (n = 40) were 14.3% and 85%, respectively. The median of EtG concentration between these two groups were significantly different. Sensitivity and specificity were both more than 90% when EtG concentrations of high-risk subjects were compared with the 30 pg/mg cutoff recommended by the Society of Hair Testing (SoHT) for diagnosing chronic excessive alcohol consumption, based on an average ethanol daily intake greater than 60 g., Conclusion: The in-house protocol for EtG analysis in hair was validated according to international standards. The protocol is a useful tool for evaluating risk for chronic excessive drinking as defined by AUDIT scores. It strongly predicted the highest level of risk, although it was inadequate for assessing lower levels of risk.