Purpose: A facile method was established to enzymatically synthesize rhapontigenin from the glycosylated parent compound rhaponticin. A novel and simple high-performance liquid chromatographic method was developed for the determination of rhapontigenin. The assay was successfully applied to both the in vitro and in vivo metabolic kinetic study of rhapontigenin. Methods: Serum, or microsomes (0.1 mL) was precipitated with acetonitrile after addition of the internal standard, daidzein. Separation was achieved on an amylose tris 3,5 dimethylphenylcarbamate column (150 × 4.6mm, ID, 5μm) with UV detection at 324nm. Hep G2 hepatoma cells were treated with rhapontigenin or rhaponticin (0-250 μg/mL) and cell viability was measured. Results: The calibration curves were linear ranging from 0.5 to 100 μg/mL. The mean extraction efficiency was > 99%. Precision of the assay (coefficient of variation) was <5%, including the limit of quantitation (0.5 μg/mL). Bias of the assay was lower than 5%. The limit of detection was 100 ng/mL for a 0.1 mL sample. One glucuronidated metabolite of rhapontigenin has been identified. Preliminary pharmacokinetic data revealed the presence of a glucuronidated metabolite in the serum and a terminal elimination t1/2 of∼6h. Rhapontigenin demonstrated concentration-dependent anti-cancer activity with an IC50115 μg/mL in HEP G2 cells while rhaponticin showed no activity across the concentrations tested in vitro. Conclusions: The preparative enzymatic synthesis method has demonstrated utility to provide sufficient rhapontigenin for pharmaceutical studies. Rhapontigenin is an active anti-cancer compound. The developed HPLC assay is sensitive, reproducible and accurate and can be applied to pharmacokinetic and metabolism studies. Copyright © 1998 by the Canadian Society for Pharmaceutical Sciences.