A liquid chromatographic procedure with electrospray ionization tandem mass spectrometric detection has been developed and validated for LSD and iso-LSD determination. A one-step liquid–liquid extraction on 1 ml blood or urine was used. The lower limit for quantitative determination was 0.02 μg/l for LSD and iso-LSD. The analytical procedure has been applied in two positive cases (case 1: LSD=0.31 μg/l, iso-LSD=0.27 μg/l in plasma and LSD=1.30 μg/l, iso-LSD=0.82 μg/l in urine; case 2: LSD=0.24 μg/l, iso-LSD=0.6 μg/l in urine). LSD metabolism was investigated using MS–MS neutral loss monitoring for the screening of potential metabolites. The main metabolite was 2-oxo-3-hydroxy-LSD (O–H–LSD) present in urine at the concentrations of 2.5 μg/l and 6.6 μg/l, respectively, for case 1 and 2, and was not present in plasma. Nor-LSD was also found in urine at 0.15 and 0.01 μg/l levels. Nor-iso-LSD, lysergic acid ethylamide (LAE), trioxylated-LSD, lysergic acid ethyl-2-hydroxyethylamide (LEO) and 13 and 14-hydroxy-LSD and their glucuronide conjugates were detected in urine using specific MS–MS transitions.