Ethyl sulfate (EtS)—a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others—was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M − H] − m/z 125 and product ions m/z 97 [HSO 4 ] − and m/z 80 [SO 3 ] − . Pentadeuterated EtS (D 5 -EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M − H] − m/z 130, product ions m/z 98 [DSO4] − and m/z 80 [SO3] − ). After addition of D 5 -EtS and D 5 -EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers’ urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs.