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Ethyl glucuronide (EtG) quantification in hair was assessed using quality controls prepared by three methods: (a) spiking hair samples with known concentrations of EtG, (b) fortifying hair by incubation of blank hair with EtG for several days or (c) use of authentic hair samples positive for EtG. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed on a Shimadzu model 8030 instrument and validated for the quantification of EtG. For two concentration levels, approximately 50 and 500pg/mg QCs, EtG concentrations were measured in duplicate (N=2) on 8 days (N=16) and intra-assay precision (repeatability) and inter-assay precision determined using one-way analysis of variance. EtG concentrations measured in authentic hair exhibited poor intra-assay precision, with coefficients of variation of 25.1 and 20.9%, compared with 17.7 and 18.5% for fortified hair and 17.4 and 11.3% for spiked hair, for the lower and higher concentrations respectively. The inter-assay precision for authentic hair was also poorer, 35.7 and 22.5%, compared with fortified (28.2 and 19.8%) and spiked (18.4 and 13.2%) hair for the lower and higher concentrations. Although spiked QCs resulted in a better repeatability and inter-assay precision, the values obtained for QCs prepared from fortified and authentic hair are likely to be more representative of case specimens. These results have implications on the interpretation of EtG concentrations when spiked QCs are used to validate methods.