A novel, robust, and stability-indicating RP-HPLC method was developed and validated for the simultaneous estimation of Sulopenem Etzadroxil and Probenecid in bulk and pharmaceutical dosage form, following the Quality by Design (QbD) approach. The study aimed to apply systematic risk assessment and statistical optimization to enhance method reliability and analytical performance. Chromatographic separation was achieved using a SunFire C18 column (250 × 4.6 mm, 5 µm) with a mobile phase of Methanol and Orthophosphoric acid (30:70 v/v) at a flow rate of 1.0 mL/min, with detection at 270 nm. The method optimization was carried out using Central Composite Design (CCD), evaluating the effects of flow rate, mobile phase ratio, and column temperature on critical quality attributes such as retention time, resolution, and theoretical plates. Statistical analysis confirmed model significance with R² values exceeding 0.99, demonstrating strong predictive capability. Validation as per ICH Q2(R2) guidelines established specificity, precision (%RSD < 2%), accuracy (recovery 98–102%), linearity (25–150 µg/mL), robustness, and sensitivity (LOD: 0.48 µg/mL; LOQ: 1.45 µg/mL). Forced degradation studies under acid, alkali, oxidative, thermal, photolytic, and neutral conditions confirmed the stability-indicating nature of the method. The optimized conditions achieved a Derringer desirability value of 1.0, signifying ideal performance. The proposed method is simple, accurate, precise, and reliable for routine quality control and stability testing of Sulopenem Etzadroxil and Probenecid in combined dosage forms, supporting regulatory compliance and lifecycle management.