The characterization of complex mixtures is a challenging issue for the development of innovative processes dedicated to biofuels and bio-products production. The huge number of compounds present in biomass fast pyrolysis oils combined with the large diversity of chemical functions represent a bottleneck as regards analytical technique development. For the extensive characterization of complex samples, supercritical fluid chromatography (SFC) can be alternative to usual separation techniques such as gas (GC) or liquid chromatography (LC). In this study, an approach is proposed to define the best conditions for the SFC separation of a fast pyrolysis bio-oil. This approach was based on SFC data obtained directly from the bio-oil itself instead of selecting model compounds as usually done. SFC conditions were optimized by using three specific, easy-to-use and quantitative criteria aiming at maximizing the separation power. Polar stationary phases (ethylpyridine bonded silica) associated to a mix of acetonitrile and water as polarity modifier provided the best results, with more than 120 peaks detected in SFC-UV.