Environmental changes and fluctuated storage conditions are the main markers for API degradation and drug-drug interactions. Avanafil (Ava), phosphodiesterase inhibitor and dapoxetine (Dap), selective serotonin reuptake inhibitor helps to treat erectile dysfunction and increase ejaculation retention. The aim of the current study is to investigate the best chromatographic condition for the determination of Ava, Dap and their degradation products at different stress conditions and develop a sensitive, rapid, and reproducible method for their simultaneous estimation either in their combined dosage form or in rat plasma. The chromatographic conditions involve separation with RP-Eclipse-XDP C18 column, (25 cm ×4.6 mm, 5µ) column at 239 nm detection wavelength and mobile phase consisting of buffer (phosphate, pH 3) to acetonitrile with ratio 55:45 (v/v) and the injection volume was 20 µl. The method was subsequently used to evaluate the mixture of Ava and Dap pharmacokinetics in rat plasma. The pharmacokinetic parameters Cmax, Tmax, t1/2, AUC0- 24, AUCtotal, Ke, Vd, and CL of the cited drugs after oral administration was determined. The obtained data demonstrate that the method was applied successfully for separating each component and their degradation products with all accepted validation parameters and the method can be applied for preclinical pharmacokinetic investigation.