A RP HPLC method for estimation of sildenafil citrate (SC) in tablet dosage form and seminal fluid was developed and validated. Best resolution was obtained with column Waters Spherisorb® C18 bonded silica, (5 µm, 4.6 x 250 mm) at 230 nm with retention time of 5.01 min. The mobile phase used was TEA (0.2%) pH adjusted at 3 with OPA and ACN (60:40) with flow rate of 1.0 mL/min. The method for estimation of sildenafil citrate in tablet dosage form was found to be linear, accurate, precise, sensitive and selective. Whereas the bioanalytical estimation of SC in seminal fluid was found to be in the range of 100 ng/mL to 10µg/mL. Method was found to be highly sensitive as LOD and LOQ were found to 0.3 µg/ml and 0.9µg/ml. The repeatability and reproducibility were within the range i.e. less than 2%.The accuracy of the method was 99.3%. The percentage purity was calculated for market formulation was 102.8%. The internal standard used for bioanalytical methods was Diclofenac sodium. The drug was extracted from seminal fluid by protein precipitation using ACN as precipitating agent. The linearity range was from 100.0ng/mL to 2.0µg/mL. The LOD and LOQ were found to 0.03µg/mL and 0.1µg/mL. The repeatability and reproducibility were within the range i.e. % RSD less than 15%. The accuracy of the method was 90.36%.and the extraction efficiency was found to be 98.25%. The stability of drug was found to be within the range i.e. less the 15%.