A simple and sensitive HPLC method using UV detection was developed to determine the concentration of protoapigenone in rat plasma. Chromatographic separation was conducted on a C-18 column with a mobile phase consisting of an acetonitrile-methanol-aqueous phase (containing 0.2% acetic acid, pH 3.0) system at a flow rate of 1.0 mL/min. The UV detector was set at 248 nm. The calibration curve was linear over the range of 0.031-10.0 mu g/mL. The lower limit of quantification was 31 ng/mL. The recoveries for plasma samples ranged from 70.3 to 82.5%. The intra- and inter-day accuracy and precision fulfilled the international standards. This method was successfully applied to a pharmacokinetic study of protoapigenone in rats after oral administration of protoapigenone. It was shown that protoapigenone could be absorbed rapidly after oral administration and could reach the maximum concentration within 1 h. Copyright (c) 2013 John Wiley & Sons, Ltd.