A simple, sensitive, selective and reproducible method based on a reversed-phase chromatography was developed for the determination of Praziquantel in rat plasma using internal standard as Diazepam. Praziquantel & Diazepam was separated on a C18 column Enable (250 mm × 4.6 mm, 5 µm particle size: Spinco Biotech Pvt Ltd), with retention times of 6.4 & 8.5 min. Ultraviolet detection was set at 225 nm. The mobile phase consisted of acetonitrile and water (60:40, v/v), running through the column at a flow rate of 1.0 mL min^-1. The chromatographic analysis was operated at an ambient temperature. Sample preparation (200 µl plasma) was done by protein precipitation using perchloric acid. Calibration curve in plasma was plotted at different concentration level 5, 50, 100, 500, 1000, 2000, 3000 & 5000 ng mL^-1 were found to be linear with correlation coefficients (r) is 0.9989. The precision of the method based on within-day repeatability and reproducibility (day-to-day variation) was within 15% (relative standard deviation: R.S.D. should be less than 15 according to CDER guidance for Bio-analytical Method Validation). Good accuracy was observed for both the intra-day or inter-day assays, as indicated by the minimal deviation of mean values. Limit of quantitation (LOQ) was accepted as 5 ng mL^-1 using 200 µl samples. The mean recovery was found to be greater than 90% for praziquantel. The method appears to be robust and has been applied for pharmacokinetic study of praziquantel, in three different groups of rats following a single oral dose of 40 mg kg^-1 body weight of praziquantel.