Quality by Design (QbD) is a scientific, risk-based strategy for systematically understanding and controlling key variables to improve reproducibility and reliability in pharmaceutical development, and its concepts have been extended to analytical method development (Analytical QbD). In this study, a QbD-based HPLC assay method for the quantification of an active pharmaceutical ingredient (API) was developed using the Fusion QbD software and verified through validation. For method optimization, key chromatographic conditions—column type, column temperature, mobile phase type, and mobile phase composition—were varied and comparatively evaluated using peak shape, retention time, tailing factor, and theoretical plates as performance metrics. Fusion QbD was also used to assess robustness by examining method performance within the selected operating ranges. The method was validated in accordance with ICH guidelines, demonstrating specificity, system suitability, linearity, and accuracy. The limit of detection (LOD) and limit of quantification (LOQ) were also determined. Overall, the QbD-based approach facilitated systematic and efficient method development, and ICH-based validation confirmed that the established HPLC assay method is suitable for API quantification.