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Abstract: The present study aims at developing a simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of pantoprazole sodium (PS) in human plasma using pantoprazole D3 (PSD3) as internal standard (IS). Chromatographic separation was performed on Zorbax SB-C18, 4.6mm×75mm, 3.5μm, 80Å column with an isocratic mobile phase composed of 10mM ammonium acetate (pH 7.10): acetonitrile (30:70, v/v), pumped at 0.6mL/min. PS and PSD3 were detected with proton adducts at m/z 384.2→200.1 and 387.1→203.1 in multiple reaction monitoring (MRM) positive mode, respectively. Precipitation method was employed in the extraction of PS and PSD3 from the biological matrix. This method was validated over a linear concentration range of 10.00–3000.00ng/mL with correlation coefficient (r)≥0.9997. Intra- and inter-day precision of PS were found to be within the range of 1.13–1.54 and 1.76–2.86, respectively. Both analytes were stable throughout freeze/thaw cycles, bench top and postoperative stability studies. This method was successfully utilized in the analysis of blood samples following oral administration of PS (40mg) in healthy human volunteers. [Copyright &y& Elsevier]