An accurate and reproducible gas chromatography-mass spectrometry (GC-MS) analytical method was developed to enable the Laboratory to determine the presence and concentration of the 2-oxo-3-hydroxy metabolite of lysergic acid diethylamide (OH-LSD) in urine. The limit of detection was 0.5 ng/mL, with a limit of quantitation established as 1.0 ng/mL. The assay was reproducible and linear over the concentration range 0.5–50.0 ng/mL, with a typical correlation coefficient (r2) of 0.997. This method was subsequently applied to the analysis of specimens submitted for routine drugs of abuse screening as part of an audit into the prevalence of LSD abuse in the West Midlands, U.K. This initial pilot study was performed using urine specimens obtained from 600 suspected LSD abusers. Following immunoassay screening, only 25 (6 female and 19 male subjects) were found to be LSD positive, but only 11 were confirmed as containing OH-LSD by GC-MS. The concentrations determined in these specimens ranged from 1.7 to 55.8 ng/mL (mean 11.0, median 5.4). This rapid and sensitive technique should enable the Laboratory to perform future audits of LSD abuse throughout the West Midlands and facilitate differential diagnoses to be made between drug misuse and underlying organic disorders.