The bioanalytical applications of supercritical fluid techniques, such as supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC), are of increasing interest. The main role of these techniques is in the sample preparation and separation of biologically active compounds, particularly drugs and their metabolites, as well as endogenous compounds. An insight is given into the different types of extracting fluids and modifiers, detectors, stationary phases, mobile phases and collection strategies. A critical discussion is presented on the existing state of the art concerning the applications of SFC and SFE with a specific focus on its advantages and limitations in the bioanalytical field. New developments and the possibilities for routine work in the near future are also covered.