The detection of LSD use continues to be a challenge for toxicology laboratories due to the very low concentrations of LSD and its metabolites in body fluids. However, significant progress has been made in the development of more sensitive and specific analytical methods. Techniques that have proven particularly effective include: (1) immunoaffinity extraction, (2) gas chromatography coupled with chemical ionization and tandem mass spectrometric detection, and (3) liquid chromatography in combination with electrospray ionization and either single-stage or tandem mass spectrometric detection. In addition, a major metabolite of LSD, 2-oxo-3-hydroxy-LSD, has been identified and found to be present in far higher concentrations than LSD in most LSD-positive urine samples.