A method of analysis of naringenin [(+/-)-4′,5,7-trihydroxyflavanone] in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism, tissue distribution in fruits and humans. A simple high-performance liquid chromatographic method was developed for simultaneous determination of naringenin enantiomers in rat and human urine. Urine (0.1 ml) was precipitated with cold acetonitrile after addition of the internal standard, daidzein. Separation was achieved on a Chiralcel OD-RH column with UV detection at 292 nm. The calibration curves were linear ranging from 0.5 to 100 μg/ml for each enantiomer. The mean extraction efficiency was >99%. Precision of the assay was <9.4% (CV), and was within 5.4% at the limit of quantitation (0.5 μg/ml). Bias of the assay was lower than 16%, and was within 15% at the limit of quantitation. The assay was applied successfully to the urinary excretion of naringenin in rats and humans. © 2005 Elsevier B.V. All rights reserved.