A stereospecific method of analysis of eriodictyol [5,7,3′,4′-tetrahydroxyflavanone] in biological fluids is necessary to study the kinetics of in vitro and in vivo metabolism, and tissue distribution in fruits and humans. A simple high-performance liquid chromatographic method was developed for the stereospecific determination of eriodictyol in rat and human urine. Separation was achieved on a Chiralpak OJ-RH column with UV detection at 288 nm. The stereospecific calibration curves were linear ranging from 0.5 to 100 μg/ml. The mean extraction efficiency was >98.8%. Precision of the assay was <15% (CV), and was within 12% at the limit of quantitation (0.5 μg/ml). Bias of the assay was lower than 8%, and was within 6% at the limit of quantitation. The assay was applied successfully to the urinary excretion of eriodictyol in rats and humans, and to the stereospecific quantification of eriodictyol in raw lemon juice, conventional and organic lemonade. © 2006 Elsevier B.V. All rights reserved.