Abstract: A high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of nicotine and cotinine enantiomers in rat plasma was developed and applied in a stereoselective pharmacokinetic study. The analytes in the rat plasma were extracted by one-step protein precipitation with methanol, then separated on a Chiralpak IG-3 column (250 mm×4.6 mm×3 μm) using a mobile phase of 0.2% ammonium formate in methanol at a flow rate of 1.2 mL/min, and finally quantified with isotope internal standard method. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode (MRM) at positive electrospray ionization interface. The results showed that nicotine and cotinine enantiomers were separated by baseline with the limit of quantitation (LOQ) of 0.5 μg/L. Parameters of method validation including the linearity, selectivity, accuracy, precision, matrix effect and stability were evaluated. The intra-day and inter-day precision and accuracy at low, medium and high concentration levels exhibited relative standard deviations (RSD) less than 12% and the accuracy ranged from 91.3% to 110.4%. This method is convenient, rapid and suitable for pharmacokinetic study of nicotine enantiomers. The main pharmacokinetic parameters including T_max, C_max, t_1/2 and AUC were calculated by noncompartment model. After subcutaneous injection of (R,S)-nicotine in rats, pharmacokinetic results suggested that no difference between nicotine enantiomers existed. Metabolic rate of cotinine enantiomers from nicotine was similar, however, the clearance rate of S-cotinine was lower than its antipodes. This study will provide valuable information for further stereoselective pharmacokinetic study and application of nicotine enantiomers.