A method of analysis of pterostilbene [trans-3,5-dimethoxy-4′-hydroxystilbene] is necessary to study the kinetics of in vitro and in vivo metabolism and determine its concentration in foodstuffs. A novel and simple high-performance liquid chromatographic method was developed for determination of pterostilbene in rat serum. Serum proteins (0.1 mL) are precipitated with cold acetonitrile after addition of the internal standard, pinosylvin. Separation was achieved on a Phenomenex C18 column (250 mm × 4.60 mm) with fluorescence excitation at 330 nm and emission at 374 nm. The calibration curves were linear ranging from 0.5 to 100 μg/mL. The mean extraction efficiency was >99%. Precision of the assay was <15% (CV), and was within 14% at the limit of quantitation (0.5 μg/mL). Bias of the assay was lower than 14%, and was within 9% at the limit of quantitation. The assay was applied successfully to the study of pterostilbene pharmacokinetics in rats. © 2006 Elsevier B.V. All rights reserved.