The interest on supercritical fluid chromatography (SFC) has considerably increased during the last decade due to the commercialization of new state-of-the-art instrumentation. Possible advantages of SFC over other chromatographic techniques are numerous: higher efficiency, faster analysis, green chemistry. The purpose of this thesis work was to evaluate the potential of modern SFC as an alternative technique to liquid chromatography in the field of pharmaceutical analysis. In a first part, some fundamental aspects of SFC, such as stationary phase evaluation, retention time modeling, sample diluent and method validation, were studied. In a second part, the technique was hyphenated with mass spectrometry (MS) and applied to the analysis of biological fluids (urine and plasma). A special focus on the field of anti-doping control was carried out and the technique showed interesting results, especially with the reduction of matrix effects.