Arecoline is the primary active and toxic constituent of areca nut. Arecaidine and arecoline N-oxide are two major active metabolites of arecoline. In this work, an accurate and simple high performance liquid chromatography tandem mass spectrometry method for simultaneous quantification of arecoline, arecaidine and arecoline N-oxide in rat plasma was developed and fully validated to study their pharmacokinetic behaviors in rats. After extracted from rat plasma by protein precipitation with methanol and then concentrated, the analytes were chromatographic separated on a Sepax Sapphire C18 analytical column. The mobile phase consisted of methanol and 2mM ammonium acetate buffer solution containing 0.2% (v/v) formic acid (8:92, v/v) under isocratic elution. The analytes were detected by multiple reaction monitoring (MRM) with an electrospray ionization source in the positive ion mode. The transitions of m/z 156.2→53.2,m/z 142.2→44.2 and m/z 172.2→60.2 were selected for arecoline, arecaidine and arecoline N-oxide, respectively. The method was linear over the concentration range of 0.5–100ng/mL for arecoline, 5–5000ng/mL for arecaidine and arecoline N-oxide with no carry-over effect. The accuracies and intra- and inter-batch precisions were all within the acceptance limits. No matrix effect and potential interconversion between the analytes and other metabolites were observed in this method. The validated method was further employed to a preclinical pharmacokinetic study of arecoline, arecaidine and arecoline N-oxide after oral treatment with 20mg/kg arecoline to rats.