Objectives: Ethyl- β -D-6-glucuronide (EtG) is a minor phase-II metabolite of ethanol. The aim of this work was to develop and validate a gas chromatography negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) method to measure EtG levels in human urine and serum with both high sensitivity and specificity. Methods: EtG was extracted and purified from 1 mL urine or 0.5 mL serum by solid-phase extraction (SPE) using Mixed-mode Anion-eXchange (Oasis®MAX) extraction cartridges, followed by derivatization with pentafluoropropionic anhydride (PFPA). The analysis was performed in the multiple reaction monitoring (MRM) mode using the transitions m/z 496→163 (for EtG quantification), m/z 347→163 and m/z 496→119 (for identification), and m/z 501→163 for the internal standard EtG-D5. The validation procedure was performed according to the guidelines of the French Society of Analytical Toxicology (SFTA) and the French Committee of Accreditation (COFRAC; LAB GTA 04). Results: Calibration curves were linear in the concentration range of 10 to 10 000 ng/mL and 5 to 1 000 ng/mL in urine and serum, respectively, with a coefficient of correlation ( r ) above 0.996. The LOD and LOQ values were 5 and 10 ng/mL, respectively, for both matrices. The intra- and inter-day precision (relative standard deviation RSD%) and relative bias were less than 20%. Conclusion: To our knowledge, this is the first report of the application of a GC-MS/MS method for EtG measurement in urine and serum. The LOQ achieved appears to be better than those reported in the literature using other validated analytical techniques. This method could be used routinely for EtG measurement in various clinical and forensic contexts.