Toxicological examination of fatal aviation accident victims routinely includes analysis of ethanol levels. However, distinguishing between antemortem ingestion and postmortem microbial formation complicates all positive ethanol results. Development of a single analytical approach to determine concentrations of 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA), two well-known metabolites of serotonin, has provided a convenient, rapid and reliable solution to this problem. Antemortem ethanol leads to an elevation in the 5-HTOL/5-HIAA ratio for 11-19 h after acute ingestion. The liquid-liquid extracts of postmortem urine samples were subjected to liquid chromatography-mass spectrometry (LC-MS) for the simultaneous quantitation of these two analytes, yielding detection limits of 0.1 ng/ml for each. Examination of the 5-HTOL/5-HIAA ratio was undertaken for 44 urine samples known to be antemortem ethanol-positive or antemortem ethanol-negative. Recent ethanol ingestion was conveniently and accurately separated using a 5-HTOL/5-HIAA ratio of 15 pmol/nmol, a value previously suggested using human volunteers. All 21 ethanol-negative postmortem samples were below this cutoff, while all 23 ethanol-positive postmortem samples were above this cutoff. Thus, we recommend the employment of this cutoff value, established using this straightforward LC-MS procedure, to confirm or deny recent antemortem ethanol ingestion in postmortem urine samples.