The behavior of ion-exchange chromatography is well understood with respect to change in ionic strength, pH, resin ligand density, bed height, elution flow rate, and gradient slope. Their relative importance for any specific chromatographic situation varies. When a chromatographic operation utilized to purify a human therapeutic protein is prepared for validation before commercial production, numerous tests have to be performed to establish the relative importance of each operating parameter to define its future role and importance in the framework of in-process controls. This prioritization process is usually performed using a purely empirical approach. In this work, we demonstrate the application of a rational approach based on chromatographic theory to prioritize operating parameters. Both methodologies, empirical and rational, were performed to evaluate a specific ion-exchange chromatography operation for the preparative separation of closely related protein species. We show that the application of the rational approach has the potential to accelerate the evaluation and significantly reduce the amount of analytical testing needed.