Specimens from fatal aviation accident victims are submitted to the FAA Civil Aerospace Medical Institute for toxicological analysis. During toxicological evaluations, ethanol analysis is performed on all cases. Care must be taken when interpreting a positive ethanol result due to the potential for postmortem ethanol formation. Several indicators of postmortem ethanol formation exist; however, none are completely reliable. The consumption of ethanol has been shown to alter the concentration of two major serotonin metabolites, 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). While the 5-HTOL/5-HIAA ratio is normally very low, previous studies using living subjects have demonstrated that the urinary 5-HTOL/5-HIAA ratio is significantly elevated for 11–19 h after acute ethanol ingestion. Recently, our laboratory developed and validated an analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a simple liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. In this previous work a 15 pmol/nmol serotonin metabolite ratio cutoff was established in postmortem urine, below which it could be conclusively determined that no recent antemortem ethanol consumption had occurred. In the current study this newly validated analytical method was applied to five ethanol-positive aviation fatalities where the origin of the ethanol present could not previously be conclusively determined. In four of the five cases examined the detected ethanol was demonstrated to be present due to postmortem microbial formation, and not consumption, even though some indication of ethanol consumption may have been present.