Testing for drugs of abuse has become commonplace and is used for a variety of indications. Commonly employed testing methods include immunoassay and chromatography. Testing methods vary in their sensitivity, specificity, time, and cost. While urine remains the most common body fluid used for testing of drugs of abuse, over the last several decades the use of alternative matrices such as blood, sweat, oral fluids, and hair has increased dramatically. Each biological matrix offers advantages and disadvantages for drug testing, and the most appropriate matrix frequently depends on the indications for the drug test. Drugs of abuse that are most commonly tested include alcohol, amphetamines, cannabinoids, cocaine, opiates, and phencyclidine. Testing may involve detection of the parent compound or metabolites and sensitivity, specificity, and reliability of drug testing may vary depending on the drug being tested. Toxicologists have a responsibility to understand the strengths and limitations of testing techniques and matrices to be able to critically evaluate the results of a drug test.