An assay has been developed for quantitative determination of lysergic acid diethylamide (LSD) and a major metabolite of LSD in human urine at concentrations as low as 10 pg/mL. In most LSD-positive urine samples the metabolite, 2-oxo-3-hydroxy-LSD, is present at higher concentrations than LSD and can be detected for a longer time than LSD after ingestion of the drug. Urine samples are extracted using Varian Bond Elut Certify extraction cartridges. Confirmatory identification is accomplished by trimethylsilylation of LSD and 2-oxo-3-hydroxy-LSD, followed by gas chromatography-tandem mass spectrometry analysis using positive ion chemical ionization and selected reaction monitoring. Commercially available lysergic acid methylpropylamide and 2-oxo-3-hydroxy-LAMPA are used as internal standards. With selected reaction monitoring, both compounds gave linear calibration curves from 10 pg/mL to 5000 pg/mL. Forty-nine human urine samples that had previously been shown to contain LSD were reanalyzed by the new method. These samples showed an average LSD concentration of 357 pg/mL and an average 2-oxo-3-hydroxy-LSD concentration of 3470 pg/mL. Additional experiments using clinical samples in which two subjects were dosed with LSD support the conclusion that analysis for 2-oxo-3-hydroxy-LSD can permit identification of LSD users for a longer period following ingestion than analysis for the parent drug.