A gradient High performance liquid chromatography (HPLC) method with UV detector was developed for the determination of drug related substances and which find their origin during the manufacture of the active pharmaceutical ingredients like, Omeprazole, Rabeprazole and Pantoprazole. The selection of these active pharmaceuticals was based on usage estimates. Such drug related substances in effluents are of increasing concern in the regulatory realm due to their potential therapeutic action in humans. A validated HPLC method was developed for the study of the selected drug related substances. Good chromatographic separation was achieved for all the substances owing to their structural similarities thereby resulting in acceptable resolution. The HPLC method employs market available Xterra RP 18 column. The constitution of the mobile phase solutions A and B are as follows: The solution A contains 10 mM Ammonium acetate buffer with pH 8 and solution B contains pure Acetonitrile. The detection wavelength used is 210 nm. The resolution between the closely eluting analytes, Pantoprazole N-Oxide and Rabeprazole N-Oxide was found to be greater than 2. The limit of quantification (LOQ) and Limit of detection (LOD) of the proposed method is in the range of 0.16 ppm – 0.3 ppm and 0.053 ppm – 0.1 ppm respectively. Recovery at limit of quantification of the pharmaceuticals were higher than 80% and the precision at limit of quantification calculated as relative standard deviation (RSD), ranged from 7.13% to 14.76%. The validated HPLC method is further employed to study the concentrations of these drug related substances in effluents pre and post treatment.