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Lysergic acid diethylamide (LSD) is a powerful hallucinogen. Its detection is limited by its low dosage; moreover, LSD is rapidly metabolized into 2-oxo-3-hydroxy-LSD (O-H-LSD). In this study we validated two methods for determination of LSD and O-H-LSD in blood. Method #1 consisted in the upgrade of a previously developed procedure for detection of 163 compounds. Method #2 was specific for LSD and O-H-LSD. Analyses were performed through LC-MS/MS by dynamic (#1) and/or MRM mode (#2), in positive ionization. Transitions were: 324→223,208 m/z for LSD; 356→237,222 m/z for O-H-LSD. Validations were performed following the AAFS’s guidelines. Linearity was good for both methods. Sensitivity was in line with previously validated methods with LOQs at 0.0375 (#1) and 0.025 (#2) ng/mL for LSD and 0.01875 (#1) and 0.0125 (#2) ng/mL for O-H-LSD. Bias and %CV always met the acceptance criteria. RRs were >83%, except for O-H-LSD with method #1. The methods were successfully applied to two real cases. Method #1 proved to be useful for screening purposes, while method #2 can represent a sensitive and reliable tool for confirmation procedures.