As more concern arises day by day for the environmental pollution due to pharmaceutical products and their toxic nature, analytical methods require characterizing drug substances and drug product composition during all phases of environmental habitats, mainly in water habitats. A simple, sensitive, and accurate analysis has been developed to estimate esomeprazole in pharmaceutical effluents, which are released from the pharmaceutical industries into the aquatic environment, by using RP-HPLC with UV detection. The developed method is highly reproducible and sensitive to determine the esomeprazole at less than 10 ppm level. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the estimation of esomeprazole in effluents or pharmaceutical industry washouts. The separation was achieved on the C18 Gemini NX column (150 mm × 4.6 mm id., 5.0 μm). Detection was carried out using a UV detector at 302 nm. The total chromatographic analysis time per sample was about 10.0 min with esomeprazole eluting at a retention time of about 5.0 min. The method was validated for accuracy, precision, specificity, linearity and sensitivity.