Solid-phase microextraction (SPME) has gained widespread acceptance for analyte-matrix separation and preconcentration. SPME is a simple, effective adsorption/desorption technique that eliminates the need for solvents or complicated apparatus for concentrating volatile or non-volatile compounds in liquid samples or headspace. SPME is compatible with analyte separation/detection by gas chromatography and high performance liquid chromatography and provides linear results for a wide range of concentrations of analytes. By controlling the polarity and thickness of the coating on the fiber, maintaining consistent sampling time, and adjusting several other extraction parameters, an analyst can ensure highly reliable results for low concentrations of analytes. This review provides updated information on SPME with chromatographic separation for the extraction and measurement of different analytes in biological fluids and materials. Firstly the background to the technique is given in terms of apparatus, fibers used, extraction conditions and derivatisation procedures. Then the different matrices, urine, blood, breast milk, hair and saliva are considered separately. Finally, the future potential of SPME for the analysis of biological samples in terms of the development of new devices and fiber chemistries as well as applications for in vivo studies are discussed.