In the present work, we describe the fundamental and practical advantages of a new strategy to improve the resolution of very closely eluting peaks within therapeutic protein samples. This approach involves the use of multiple isocratic steps, together with the addition of a steep negative gradient segment (with a decrease in mobile phase strength) to "park" a slightly more retained peak somewhere along the column (at a given migration distance), while a slightly less retained compound can be eluted. First, some model calculations were performed to highlight the potential of this innovative approach. For this purpose, the retention parameters (logk₀ and S) for two case studies were considered, namely the analysis of a mixture of two therapeutic mAbs (simple to resolve sample) and separation of a therapeutic mAb from its main variant (challenging to resolve sample). The results confirm that the insertion of a negative segment into a multi-isocratic elution program can be a good tool to improve selectivity between critical peak pairs. However, it is also important to keep in mind that this approach only works with large solutes, which more or less follow an “on-off” type elution behavior. Two real applications were successfully developed to illustrate the practical advantage of this new approach, including the separation of a therapeutic mAb from its main variant possessing very close elution behavior, and the separation of a carrier protein from an intact mAb as might be encountered in a quantitative bioanalysis assay. These two examples demonstrate that improved selectivity can be achieved for protein RPLC through the inclusion of a negative gradient slope that selectively bifurcates the elution of two or more peaks of interest.