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A highly sensitive and rapid LC–MS/MS method was developed, fully optimized and validated for the simultaneous determination of Ledipasvir (LED) and Sofosbuvir (SOF) in the presence of its major metabolite GS-331007 in human plasma using Daclatasvir as internal standard (IS). The extraction of analytes and IS from plasma was performed using liquid-liquid extraction with ethyl acetate. The chromatographic separation of these prepared samples was achieved on Xterra MS C8 column (4.6 × 50 mm,5 μm) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.5; 10 mM), acetonitrile and methanol pumped at a flow rate 0.7 mL min−1.The detection was performed on API4000 triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) positive electrospray ionization interface. The method was validated according to FDA guidelines for bio-analytical methods with respect to linearity, accuracy, precision, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over a concentration range of 0.1–1000, 0.3–3000 and 3.0–3000 ng mL−1 for LED, SOF and GS-331007; respectively by applying weighted least-squares linear regression method (1/x2). The wider range of quantification in a shorter period of separation time less than 5.0 min allowed monitoring the serum concentration of analytes up to 144 h. The proposed method can be successfully applied for pharmacokinetic and bioequivalence studies in healthy human volunteers.A highly sensitive LC-MS/MS method was developed for analysis of Ledipasvir, sofosbuvir in the presence of its metabolite GS-331007 in human plasma.The method was fully validated according to FDA guidelines for bio-analytical methods with respect to linearity, accuracy, precision, recovery, selectivity, carry over, stability and dilution integrity.The proposed method can be applied to pharmacokinetic and bioequivalence studies in human volunteers.Our study proved that there was no significant difference in pharmacokinetic parameters with other reported papers.