This paper reviews procedures for screening, identification and quantification of drugs, poisons and their metabolites in biosamples, and the corresponding work-up procedures. Gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry are mostly used today in analytical toxicology. Selection of the most appropriate biosample, e.g., ante/postmortem blood, urine, or tissues or alternative matrices like hair, sweat and oral fluid, nails or meconium, is discussed. The importance of quality control and possibilities and limitations of interpretation of the analytical result are also discussed.