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A simple and reliable LC-ESI-MS method for the determination of peimine and peiminine in rat plasma was developed for the first time. The method was proven to be specific and sensitive by carrying out validation. The analytes were extracted from rat plasma via solid-phase extraction on Waters Oasis MCX cartridges. Chromatography separation was achieved on a C-18 column using 10 mm ammonium acetate (adjusted to pH 3.0 with glacial acetic acid)-acetonitrile (85:15, v/v) as mobile phase. The linear range was 1-100 ng/mL for peimine and peiminine. Intra- and inter-day precisiond were less than 10%. Accuracies were within 85-115% of their nominal concentrations. The limit of quantification was 1 ng/mL for both analytes. The developed assay was successfully applied to pharmacokinetic study of peimine and peiminine in rats orally administered the alkaloids extracts from Bulbus Fritillariae, demonstrating a possible broader spectrum of applications of this method. Copyright (C) 2009 John Wiley & Sons, Ltd.