A method of analysis for 3-methoxypterostilbene [trans-3,3′5-trimethoxy-4′hydroxystilbene] in biological fluids is necessary to study pharmacokinetics. A novel and simple high-performance liquid chromatographic method was developed for the determination of 3-methoxypterostilbene in rat serum and urine. The internal standard, pinosylvin, was added to 0.1 mL serum or urine (serum proteins were precipitated with cold acetonitrile at −20°C). Separation was achieved with a Phenomenex® C18 (2) (5 µm, 250 × 4.60 mm) column with ultraviolet detection at 327 nm. The calibration curves in both matrices were linear ranging from 0.05 to 100 µg/mL, and the mean extraction efficiency was >99%. Precision of the assay for both matrices was <12% (RSD) and was within 13% for all points on the calibration curve. The limit of quantification for this method was 0.05 µg/mL. The assay was successfully applied to a preliminary study of 3-methoxypterostilbene pharmacokinetics in a rat. Copyright © 2012 John Wiley & Sons, Ltd.