Specimens from fatal aviation accident victims are routinely submitted for toxicological analysis; ethanol analysis is performed on all cases. Since there is a potential for postmortem ethanol formation, it is crucial that care be taken in the interpretation of the results. The consumption of ethanol alters two major serotonin metabolites: 5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA). The current article describes the use of a newly developed and validated analytical method for the simultaneous determination of both 5-HTOL and 5-HIAA in forensic urine samples using a liquid/liquid extraction and LC/MS/MS and LC/MS/MS/MS. The method was applied to five ethanol-positive aviation fatalities from separate accidents where the source of ethanol could not be conclusively attributed to either antemortem ethanol consumption or postmortem microbial ethanol formation. Employing the new procedure, the results showed that four of the five cases had ethanol present due to postmortem microbial formation rather than ethanol consumption prior to death. The relative ease of use of the new procedure should make it readily applicable to the routine assessment of ethanol in postmortem fluid and tissue samples. Figure, references