Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, twodimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.