A method of analysis of hesperetin (+/ - 3,5,7-trihydroxy-4′- methoxyflavanone) in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism and tissue distribution. A simple high-performance liquid chromatographic method was developed for simultaneous determination of hesperetin enantiomers in rat serum, and rat and human urine. Serum and urine (0.1 ml) were precipitated with cold acetonitrile after addition of the internal standard, 7-methoxycoumarin. Separation was achieved on a Chiralpak AD-RH column with UV detection at 298 nm. The calibration curve was linear ranging from 0.5 to 100 μg/ml for each enantiomer. The mean extraction efficiency was >98%. Precision of the assay was <5% (CV), and was within 5% at the limit of quantitation (0.5 μg/ml). Bias of the assay was lower than 5%, and was within 5% at the limit of quantitation. The assay was applied successfully to the urinary excretion of hesperetin in rats and humans. © 2004 Elsevier B.V. All rights reserved.