Objective <p>The present research aims to establish an RP-HPLC method for the simultaneous estimation of Sulopenem etzadroxil and Probenecid in a synthetic mixture and a combined dosage form by Design of Experiment and green chemistry approach.</p> Methods <p>The Box-Behnken design with quadratic model and response surface methodology was adopted to ensure the effect of independent variables (Temperature, flow rate, and percentage of aqueous mobile phase) on the responses or dependent variables (RT of both drugs, resolution, and USP plate count of both drug peaks) to optimize the method confirmation of design space and robustness. The effective separation of Sulopenem etzadroxil and Probenecid was achieved with Interstil column C18 (250 × 2.1&#xa0;mm,1.8&#xa0;μm), 0.1% orthophosphoric acid: acetonitrile (38:62 v/v) at a flow rate of 1.0mL/min, column temperature of 32&#xa0;°C, and isocratic elution at 210&#xa0;nm. The elution of Sulopenem etzadroxil and Probenecid was noticed at 2.52 and 3.00&#xa0;min, with considerable system suitability and resolution with the optimized conditions. The greenness assessment of the eco-friendly nature of the developed method was determined by the green analytical tools like the Green Analytical Procedure Index, Analytical Greenness, and the Analytical Eco Scale.</p> Results <p>Sulopenem etzadroxil and Probenecid showed good linearity from 12.5 to 75&#xa0;µg/mL with R² of 0.999 and 0.999, respectively. The % RSD for both intraday and inter-day precision was determined to be in the range of 0.5 to 0.9. The detection and quantification limits of Sulopenem etzadroxil and Probenecid were calculated to be 0.011&#xa0;µg/mL, 0.034&#xa0;µg/mL, and 0.010&#xa0;µg/mL, 0.032&#xa0;µg/mL, respectively, by the standard deviation method. The stability-indicating property of the current method could be established by the forced degradation studies. The degradants produced by the stressed sample were distinctly separated from the peaks of Sulopenem etzadroxil and Probenecid. An analytical greenness score of 0.81 out of 1, a score of 81 out of 100 in the analytical eco scale, and 70% of green zones and 20% of yellow zones in the Green Analytical Procedure Index diagram confirm the greenness and environmental sustainability of the method.</p> Conclusion <p>The quicker elution time and excellent sensitivity of both Sulopenem etzadroxil and Probenecid, with good eco-friendly nature, make this method sustainable for regular analysis of Sulopenem etzadroxil and Probenecid.</p> Graphical Abstract <p></p>