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SFC was for the first time applied for separation of hydrophilic furostanol saponins.The methoxylation of the hydroxyl at C-22 position of furostanol saponins in the eluted process by using methanol as co-solvent was suppressed.SFC was effective in separating the similar furostanol saponins which share the same aglycone and vary in sugar chain.SFC could be applied for profiling the Chinese materia medicas which mainly contain steroidal saponins.Supercritical fluid chromatography (SFC) has good separation efficiency and is suitable for separating weakly polar compounds. Furostanol saponins, as an important kind of steroidal saponins, generally have two sugar chains, which are polar and hydrophilic. The hydroxyl group at the C-22 position of furostanol saponins is active and easily reacts with lower alcohols under appropriate conditions. The separation of hydrophilic furostanol saponins was tested by SFC in this study. The effects of chromatographic conditions on the separation of the mixed furostanol saponins and their hydroxyl derivatives at the C-22 position were studied. The conditions for SFC, which included different column polarity, modifier, additive, and column temperature, were tested. After optimization, the mixed 10 similar structures of furostanol saponins were separated in 22 min on the Diol column at a temperature of 40 °C. The mobile phase was CO2 (mobile phase A) and methanol (containing 0.2% NH3·H2O and 3% H2O) (mobile phase B). The backpressure was maintained isobarically at 11.03 MPa. SFC was found to be effective in separating the furostanol saponins that shared the same aglycone but varied in sugar chains. SFC was sensitive to the number and type of sugars. The resolution of furostanol saponin isomers was not ideal. The extract of Dioscorea zingiberensis C. H. Wright was profiled by SFC–quadrupole time-of-flight mass spectrometry. The main saponins of the extract were well separated. Therefore, SFC could be used for separating hydrophilic furostanol saponins and analyzing traditional Chinese medicines that mainly contained steroidal saponins.