A highly sensitive ultra-fast LC-MS/MS based bioanalytical method for the measurement of Rabeprazole in human plasma was developed and validated using 13C-D3-Rabeprazole as internal standard. Rabeprazole is a sulfabenzimidazole class of compounds, setting chromatography for these classes of compounds is always a challenge. Rabeprazole was extracted from human plasma samples by liquid-liquid extraction (LLE) and separated on a short reverse phase Ascentis® Express C18, 50 mm × 4.6 mm, 2.7 μm column by isocratic elution with 40% 10 mM ammonium acetate solution and 60% acetonitrile at a flow rate of 0.700 mL/min. Rabeprazole and its labeled internal standard were detected by multiple reaction monitoring (MRM) mode using electro spray ionization (ESI). All the validation parameters as per current guidelines like specificity, selectivity, accuracy, precision, recovery, matrix factor, haemolysis effect and stability are assessed in human plasma. Rabeprazole was found to be linear over a range of 0.1 ng/mL to 150 ng/mL in human plasma. LLOQ of0.1 ng/mL is sensitive enough for application to different clinical studies. The intra- and inter-day precision was less than 10% and accuracy was within -3.33 to 10.00%. Recovery (70%) was consistent across the linear dynamic range of the method. The validated method is a simple, accurate, precise and robust to measure Rabeprazole in human plasma and could be used for application to any format of clinical studies. © 2016 Shankar S Velan and Suneetha Vuppu.