In the treatment of hepatitis C, direct-acting antivirals (DAA) are highly efficient and well tolerated with a series of DAA combinations available for treatment. A sensitive high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the simultaneous quantification of Sofosbuvir (SOF) and Daclatasvir (DAC) in human plasma. Sofosbuvir D6 (SOF D6) and Daclatasvir (C2H6)-C-13-H-2 (DAC (C2H6)-C-13-H-2) are used as internalstandard (IS). Quantification for both the analytes has been attained with MS-MS detection in positive ion mode using an Acquity UPLC system (Waters) equipped with Waters Xevo TQ MS system with a Gemini NX 5 mu C18 (50 x 2.0mm) (Phenomenex) column, and a gradient mobile phase consisting 5 mM Ammonium Formate buffer: Acetonitrile at a flow rate of 0.300 mL/min is used as mobile phase to separate the analytes and detection is performed by electrospray ionization technique using the mass spectrometer. Full validation is performed for bio-analytical methods with respect to linearity, precision, accuracy, selectivity, carry-over, stability and dilution integrity. Linearity is obtained over a concentration range of 10.002 -3000.488 and 10.004 -3001.218 ng mL(-1) for SOF and DAC respectively by applying weighted least-squares linear regression method (1/x2). The developed method was applied successfully in bioequivalence and/or clinical studies in 48 male subjects for the simultaneous quantification of SOF and DAC.