Testing for illicit drug use is used in pre-employment checks, the criminal justice system, sports medicine and for screening and confirmatory purposes in patients with physical and mental illnesses. The types of drugs tested for and the methods used vary depending on the indication. This article focuses primarily on blood, urine and oral fluids, which are specimens more commonly used in mental health settings, although hair and sweat are increasingly used in medico-legal cases and in child protection issues. The main drugs and their metabolism are discussed to gain a better understanding of the methods used and for accurate interpretation. Methods to ensure validity during sample collection are explored. False-positive and false-negative tests are common and possible confounders are discussed.