A novel high pressure liquid chromatography method coupled with diode array detector (HPLC-DAD) was developed and found to be efficient, sensitive, and selective for the simultaneous analysis of Trifluridine (TRI), and its metabolites 5-trifluoromethyl uracil (TFMU) and 5-carboxydeoxyuridine (CDMU) and Tipiracil (TIP) in a spiked human plasma. The HPLC-DAD technique that developed was validated in accordance with the most recent ICH M10 criteria. Sample pre-treatment was performed using a solid phase extraction with InertSep HLB reversed phase sorbent 30 mg/ml cartridge. The compounds were analyzed by liquid chromatography UV detection on a Inertsil C18 (250 mm × 4.6 mm ID, Particle size: 5 µm) with a mobile phase containing sodium acetate buffer pH (3.8; 10 mM) - acetonitrile (70:30, v/v) with flow rate of 0.8 ml/min with UV detection at 254 nm. The method was linear in the range 200–5000 ng/ml for TRI, TFMU, CDMU, and 100–1500 ng/ml for TIP. With the exception of the lower limit of quantification, where they were within ≤ 20%. The accuracy and precision for spiked human plasma were within ≤ 15% for all concentrations.