The aim of this review is to provide a comprehensive overview of screening and confirmation methods reported to determine the low-molecular weight compound and drug of abuse gamma-hydroxybutyric acid (GHB) in biological fluids. The polar nature, the endogenous presence, rapid metabolization following ingestion and stability issues during storage (de novo formation and interconversion between GHB and its lactone form gamma-butyrolactone), impose challenges for the analyst and for the interpretation of a positive result. First, possible screening procedures for GHB are discussed, including colorimetric, enzymatic and chromatography-based procedures. Confirmation methods for clinical and forensic cases mostly involve gas chromatography (coupled to mass spectrometry), although also liquid chromatography and capillary zone electrophoresis have been applied. Prior to injection, sample preparation techniques include (a combination of) liquid-liquid, solid-phase or headspace extraction, as well as chemical modification of the polar compound. Also simple dilute-and-shoot may be sufficient in the case of urine or serum samples. Advantages, limitations and trends are discussed.