Abstract: Asparagus cochinchinensis is a commonly used traditional Chinese medicine with steroidal saponins as its main active ingredients. Due to the structural similarity and size of the steroidal saponins, these compounds cannot always be effectively separated by a combination of normal phase silica gel column chromatography and reversed phase ODS column chromatography. In this experiment, chromatographic columns with different separation mechanisms were systematically screened, and it was found that a chiral chromatographic cellulose column could effectively separate these components. This column was used to separate 3 mixtures to obtain 6 single compounds (1-6). Structural identification showed that the singular structural difference between these poorly separated components resides in a terminal glycosyl group (xylose or rhamnose) in the C-3 glycosyl chain, and compounds 4 and 6 are two new steroidal saponins. Since the structures of compounds are often unknown during the isolation and purification of natural products, chiral columns are rarely used. This study suggests that chiral chromatographic columns are a valuable option for natural products that are difficult to separate by conventional means.