Abstract: The resolution and pharmacokinetics of pantoprazole sodium chiral enantiomers in rat plasma samples were established by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The analytes and the internal standard were separated on Chiralpak IE chromatography column (4.6 mm×250 mm×5 μm) using a mobile phase of 0.1% formic acid in water and acetonitrile (30∶70, V/V) at a flow rate of 0.9 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 382.0>229.8 for pantoprazole, m/z293.9>249.6 for diclofenac sodium (IS) using negative electrospray ionization interface. The results showed that the linear range was 5.0-5 000.0 μg/L for pantoprazole, the limit of quantitation (LOQ) was 5.0 μg/L. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concen tration levels exhibited relative standard deviations (RSD) less than 10% and the accuracy ranged from 97.03% to 101.86%. Recoveries of pantoprazole enantiomers were in the range of 91.1%-114.3%. This method is sensitive and repeatable, which is suitable for pharmacokinetic studies.