ABSTRACTIn the last decade, the instrumentation improvements in supercritical fluid chromatography (SFC) and the hyphenation to mass spectrometry (MS), have increased the SFC acceptance between scientists, becoming today a valuable tool in analytical chemistry. The unique selectivity, short analysis times, low consumption of organic solvents, and the greener mobile phase, have contributed to expanding its applicability which has led to an increase in the number of publications especially in the bioanalysis area. This work reviews the advantages and main applications of SFC in bioanalysis during the last 5 years. Fundamental aspects concerning mobile phase composition, stationary phase, hyphenation to MS as well as matrix effect have been discussed. Finally, the most relevant applications have been summarized.