To evaluate the role of ethyl glucuronide (EtG) and ethyl sulphate (EtS) analysis in improving the interpretation of postmortem blood alcohol results. Postmortem ethanol formation is a classic problem in forensic toxicology. EtG and EtS are minority metabolites of ethanol, produced by non-oxidative reactions; they are sensitive and specific markers of ethanol intake that can be useful to distinguish antemortem alcohol intake from postmortem ethanol formation. A sensitive method of EtG and EtS analysis would help to improve the interpretation of questionable alcohol results. EtG and EtS were analysed in whole blood (WB) and vitreous humour (VH), if available, from 48 autopsy cases where ethanol is routinely analysed by headspace gas chromatography with flame ionization detector (GC-FID; Calibration range 0.1–4.0 g/L; LOD 0.04 g/L). WB and VH samples were subjected to protein precipitation, followed by ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS), with the multiple reaction monitoring (MRM) approach in negative mode, for EtG and EtS analysis. Calibration ranges for both analytes were 10–5000 ng/mL; samples were diluted where necessary. Based on ethanol findings and available samples, cases were classified into four groups: – G1: positive results for ethanol in WB and VH, strongly indicative of antemortem alcohol intake ( n = 20, blood alcohol concentration range: 0.1–3.1 g/L, median 1.3 g/L); – G2: no detection of ethanol in WB ( n = 8); – G3: positive results for ethanol in WB, but no detection of alcohol in VH ( n = 12, BAC range: 0.1–> 4.0 g/L, median 0.2 g/L); – G4: positive results for ethanol in WB but VH in poor condition (bloody) or no available VH ( n = 8, BAC range: 0.2–1.9 g/L, median 0.5 g/L). G1: all cases were positive in WB for EtG (range 28–28,314 ng/mL; median 1179 ng/mL) and EtS (range 31–3686 ng/mL; median 450 ng/mL). From cases with HV available for EtG and EtS analysis ( n = 16), 13 were found positive for both analites, two only for EtG and another one only for EtS. G2: EtG and EtS were no detected in WB, nor in HV, in any case. G3: in one of the cases EtG and EtS were positive in WB (131 ng/mL and 181 ng/mL, respectively; BAC 0.13 g/L) and HV. EtG alone was detected in WB and HV in one case, and only in HV in another case. None of them were detected in 9 cases in this group. G4: in two cases EtG and EtS were detected in WB (22,802 ng/mL and 2336 ng/mL, respectively; BAC 1.9 g/L; and 378 ng/mL and 104 ng/mL, respectively; BAC 0.3 g/L) and HV. EtG alone was detected only in WB in one case, and only in HV in another case. None of them were detected in the 4 cases in this group. In most cases from G3 the BAC values were less than 0.4 g/L. Cases with high BAC in this group were deceased with decompensated diabetes and sudden infant deaths; the alcohol detected in blood was probably originated postmortem through microbiological fermentation. Seven cases in G4 were corpses found 1–10 days after death, with variable degree of putrefaction and high risk of postmortem generation of alcohol. Antemortem alcohol intake could be stablished where EtS was detected, taking into account that EtG can be both produced and degraded by microorganisms. The joint analysis of EtG and EtS in postmortem blood and vitreous humour samples plays a key role in the interpretation of most cases where antemortem alcohol intake remains doubtful from the available data of blood alcohol concentration.