Sofosbuvir (SOF) and daclatasvir (DCS) are novel, recently developed direct acting antiviral agents characterized by potent anti-hepatitis C virus action. A fast and efficient HPLC-UV method was developed, validated and applied for simultaneous determination of SOF and DCS in pharmaceutical formulations and biological fluids based on coupling liquid-liquid extraction with ultrasound and dual wavelength detection at λmax; 260 and 313 nm for SOF and DCS, respectively. This approach provided simple, sensitive, specific and cost-effective determination of the SOF-DCS mixture with good recoveries of the analytes from plasma. Analytes were separated within 7 min on C18 analytical column with acetonitrile–10 mM acetate buffer of pH 5.0 at a flow rate of 1.0 mL min−1. The linear ranges were 1–20 μg mL−1 for SOF and 0.6–6 μg mL−1 for DCS with correlation coefficients ≥0.9995. The detection limits in spiked rabbit plasma were 0.20 and 0.19 μg mL−1 for SOF and DCS, respectively. The method was validated according to ICH and US-FDA guidelines. Finally, the method was successfully applied for simultaneous pharmacokinetic studies of SOF and DCS in rabbits using rofecoxib as internal standard.