AbstractDecontamination of samples prior to analysis is common practice and recommended for ethyl glucuronide (EtG) hair testing. The aim of this study was to evaluate the applied decontamination procedure during routine hair EtG analysis by monitoring the ethyl glucuronide concentrations in the washing solutions from a representative cohort of individual hair samples. Hair samples from 150 individuals were tested for hair EtG by a validated routine procedure (liquid chromatography/tandem mass spectrometry). A four‐step decontamination procedure (ethanol, water, acetone, dichloromethane) was applied to all samples prior to analysis. Hair samples from 20 individuals were analyzed along with the complete set of individual washing solutions. Hair samples from an additional 130 individuals were analyzed along with the corresponding aqueous wash fraction only. No EtG was detected in the washing solutions from hair samples that tested negative for EtG (n = 42). Hair samples positive for ethyl glucuronide (n = 108) were found to liberate different amounts of EtG during decontamination: whereas no, or low portions of, EtG (< 10% of extracted hair EtG) were found in the corresponding washing solutions of the majority (n = 91) of individual samples, there was a minority of samples (n = 6) with more than half of the extracted hair EtG present in the decontamination solvent. No correlation of the decontaminated amount of EtG and the extracted hair EtG was observed. Further experimental studies are necessary to investigate if the observed easily removable fraction of EtG is associated with external contamination and if analysis of wash solutions could be helpful for identifying external contamination in hair testing for ethyl glucuronide.