The goal of this second part was (i) to evaluate the performance of commercially available HIC columnsand (ii) to develop a fast and automated “phase system” (i.e. stationary phase and salt type) optimizationprocedure for the analytical characterization of protein biopharmaceuticals. For this purpose, varioustherapeutic mAbs (denosumab, palivizumab, pertuzumab, rituximab and bevacizumab) and a cysteinelinked ADC (brentuximab-vedotin) were selected as model substances. Several HIC column chemistries(butyl, ether and alkylamide) from different providers were evaluated in four different buffer systems(sodium acetate, sodium chloride, ammonium acetate and ammonium sulfate). As stationary phases, thehistorical TSK gel Butyl NPR phase and the brand new Thermo MAbPac HIC-10 were found to be themost versatile ones in terms of hydrophobicity, peak capacity and achievable selectivity. As salt types,ammonium sulfate and sodium acetate were found to be particularly well adapted for the analyticalcharacterization of mAbs and ADCs, but it is important to keep in mind that a concentration 2 to 3-timeshigher of sodium acetate versus ammonium sulfate is required to achieve a similar retention in HIC. Afterselection of the most appropriate phase systems, the optimization of the separation can be carried outby computer assisted retention modeling in a high throughput manner.