A simple and rapid method for preparative separation of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, and Rd from Panax notoginseng was developed with preparative high-performance liquid chromatography (prep-HPLC). The satisfied chromatographic separation of individual compounds was achieved using a mobile phase of ethanol-water system on an Alltech Alltima C18 column in less than 1 hr. The purity of all 5 saponins obtained was determined to be more than 96% by normalization of the peak area detected by HPLC-UV. Chemical structures of purified products were confirmed by MS and their reference compounds. The results indicated that this method is useful for the preparative-scale separation of notoginsenoside R1, ginsenosides Re, Rg1, Rb1, and Re in a simple, rapid, ease of use, and cost-effective manner.