Hoodia gordonii, family Asclepiadaceae, is a succulent plant and is traditionally used in southern Africa for its appetite-suppressant properties. A high-performance liquid chromatographic (HPLC) method with UV detection for analysis of 11 oxypregnane glycosides from H. gordonii has been developed. The simultaneous analysis of 11 oxypregnane glycosides was achieved with a Phenomenex (Torrance, CA) reversed-phase C18 column using gradient mobile phase of water and acetonitrile, both containing 0.025% trifluoroacetic acid. The developed method was applied to the identification of oxypregnane glycosides in 3 different species of Hoodia and 23 related genera. The HPLC profiles of various plant samples were compared for the presence of oxypregnane glycosides.