AbstractChromatographic methods (paper chromatography, thin layer chromatography, high performance liquid chromatography, gas chromatography and gas chromatography‐mass spectrometry) for the determination of clinically important steroids in biological specimens are reviewed. The emphasis is on the use of gas chromatography, gas chromatography‐mass spectrometry and high performance liquid chromatography as reference rather than routine techniques. Chromatographic methods are compared with colorimetric, fluorimetric and radioimmunoassay procedures in terms of simplicity of operation, cost and ability to analyse large numbers of specimens. The importance of correct specimen collection and storage are discussed. Sample preparation techniques for the various analytical methods are described. These include extraction of free and conjugated steroids from serum, plasma, urine and saliva by solvent partition, with polymer‐based resins such as Amberlite XAD‐2, DEAE‐Sephadex and Sephadex resins bonded with various other function groups and, more recently, with chemically bonded reversed‐ph