Background: Current guidelines for the treatment of hypertension recommend combination therapy, which intends to control blood pressure and enhance cardiovascular protection. Materials and Methods: A sensitive, reliable and selective tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous quantification of amlodipine (AML), valsartan (VAL) and hydrochlorothiazide (HCTZ) in human plasma. The chromatographic system was equipped with ACE 5 C8 (50 X 2.1 mm) column and utilized a mobile phase composition of 0.5 mM Ammonium Chloride & 0.04% FA-Methanol (45:55% v/v). The method used three internal standards; AML-D4, HCTZ-D2 C13 and VAL-D3 with 10% intra- and inter-day precision, and 6% bias for all the analytes. Results: The assay was found to be linear with R-2 > 0.998, and the limits of quantification for AML, VAL and HCTZ were 0.2, 50.0 and 2.0 ng/mL, respectively. The analytes were found to be stable in plasma samples over short and long term storage. : The developed method is rapid with a run time of 3.5 min and cost-effective since the simple sample preparation method is adopted. This method was successfully applied for the bioequivalence study of AML, VAL, and HCTZ in human plasma after administration of the fixed-dose combination tablet of (10/160/25 mg). Pharmacokinetic parameters (Cmax and AUC0-72) for AML and (Cmax, AUC0-t, AUC0-∞) for VAL and HCTZ were used for bioequivalence assessment. These were determined by noncompartmental analysis of concentration data. Conclusion: The result showed 90% confidence intervals (obtained by ANOVA) which were within the predefined ranges. As a consequence, this method can be successfully applied for measuring and quantifying a large number of samples.