A simple, sensitive and accurate analytical has been developed to estimate esomeprazole in pharmaceutical effluents, which are releasing from the pharmaceutical industries into aquatic environment by using RP-HPLC with UV detection. the developed method is highly reproducible and sensitive to determine the esomeprazole in 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 C18 Gemini NX column (150mm × 4.6mm i.d., 5.0μm) using a mixed buffer of sodium dihydrogen phosphate monohydrate and dibasic sodium phosphate anhydrous having a pH of 7.3 as buffer, and the mobile phase is a mixture of 350 mL buffer: 500 mL acetonitrile: 150 mL of water in isocratic mode as mobile phase and at a flow rate of 0.8 mL/min. 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 retention time of about 5.0 min. The method was validated for accuracy, precision, specificity, linearity, and sensitivity. Validation studies demonstrated that this HPLC method is accurate, specific, rapid, reliable, and reproducible. Linearity was observed for esomeprazole in the concentration range of 0.025–10 µg/mL (R2 > 0.95). The limit of detection (LOD) and limit of quantitation (LOQ) was found to be 0.003 µg/ml and 0.009 µg/ml respectively for esomeprazole, the method was validated as per ICH guidelines. The RSD for intra-day and inter-day precision were found to be less than 5%. The percentage recovery was in good agreement and the method is simple, specific, precise, and accurate for the determination of esomeprazole in the pharmaceutical industry washouts.