Eprosartan mesylate and hydrochlorothiazide are antihypertensive agents used in combination therapy. The objective of the present study was to develop and validate a simple, rapid and sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous determination of eprosartan mesylate and hydrochlorothiazide in human plasma. Methods: Chromatographic separation was achieved on an Agilent C18 column (150 × 4.6 mm, 5 µm) using 0.1% orthophosphoric acid (pH 2.2) and acetonitrile (60:40, v/v) as the mobile phase under isocratic conditions. The flow rate was 1.0 mL/min, the injection volume was 20 µL, the column temperature was 30 °C and detection was performed at 240 nm. Valsartan was used as the internal standard. Plasma samples were processed by liquid-liquid extraction with acetonitrile. Results: Eprosartan and hydrochlorothiazide were well resolved from the internal standard, with reported retention times of approximately 2.318, 2.713 and 3.698 min, respectively. The method was linear over 80–3200 ng/mL for eprosartan and 8.5–340 ng/mL for hydrochlorothiazide, with a reported coefficient of determination (r²) of 0.999. The reported overall mean recoveries were 98.858% for eprosartan and 98.67% for hydrochlorothiazide. Selectivity, precision, accuracy and stability results were reported to meet the stated acceptance requirements. Conclusion: The developed RP-HPLC method provides a practical analytical approach for simultaneous determination of eprosartan mesylate and hydrochlorothiazide in human plasma.