The goal of this work was to provide a technical solution for the automated optimization of multi-column systems for protein separation and fractionation. Both algorithm and a software that can be downloaded are provided. In this algorithm, the length and order of the individual column segments can be consid- ered. Various solutions are provided by the algorithm, including i) to obtain uniform peak distribution, ii) to park the different species at the inlet of the individual column segments, and iii) to elute all species as a single peak. Two representative examples are presented, showing the possibility to obtain uniform selectivity between monoclonal antibody (mAb) sub-units, and the on-column fractioning of intact mAbs.