Affinity liquid chromatography using FcRn and FcγRIIIa columns can provide important information on the drug effector functions and the unique PK/PD properties of therapeutic mAbs. In this study, we propose a unique strategy to improve the performance of affinity chromatography by applying pH-gradient programs that incorporate multi-isocratic and negative gradient segments. These alternative gradient programs are known to greatly improve the separation of large solutes that follow a “bind-and-elute” type retention behavior. First, judicious optimization of the mobile phase compositions was performed to obtain a linear pH response. Then, with the developed strategy using multi-isocratic analysis conditions, the FcRn affinity separation selectivity for the analysis of oxidized mAb species was greatly improved. Furthermore, the introduction of negative gradient segments after each eluted peak improved the resolution between multiple glycosylated mAb species on the FcγRIIIa column. Therefore, this work provides a new strategy to improve the performance of affinity chromatography with mAb species, and could assist in the development of more accurate binding assays for important critical quality attributes related to FcRn and FcγRIIIa binding.