Physalins are a novel class of natural products, having 13,14-α-16,24-cyclesteroid structures. They are potential anticancer agents, since they contain the methylene lactone system observed in many sesquiterpene lactones. Also, they have been shown to possess anti-inflammatory and anti-arthritic activities. These compounds have similar structures and hence their separation by the usual techniques is difficult. We have been successful in the separation of physalins by high-performance liquid chromatography (HPLC). The six physalins A (I), B (II), Bepoxide (III), D (5,6-dihydroxyphysalin B) (IV), E (V) and H (VI) were chosen for this work. They were found to exhibit reasonably strong UV absorption and could be detected in low concentration, using a UV detector (254 nm).