Publisher Summary This chapter discusses the separation and detection methods employed in human and animal doping control to detect the misuse of the main groups of prohibited substances: anabolic steroids, stimulants, diuretics, β-adrenergic drugs (agonists and antagonists), anti-inflammatory drugs, and peptide hormones. For all groups of drugs, a brief description on the physico-chemical properties and metabolic and excretion data for each kind of compounds, a review of analytical methodologies is described in the chapter. The misuse of drugs in human and animal sports in an attempt to enhance performance, usually known as doping, is unfortunately an extensive and old practice. Ethical and health aspects are of particular concern. Sports, fair play, medical ethics, and potential risks for the health of athletes lead to consider this practice unacceptable. Among the prohibited methods are blood doping (administration of blood, red blood cells, artificial oxygen carriers or related blood products) together with pharmacological, chemical and physical manipulation that alters the integrity and validity of urine samples.