Abstract Background Topographical modifications of the surface influence several cell functions and can be exploited to modulate cellular activities such as adhesion, migration and proliferation. These complex interactions are cell-type specific, therefore engineered substrates featuring patterns of two or more different topographies may be used to obtain the selective separation of different cell lineages. This process has the potential to enhance the performance of biomedical devices promoting, for example, the local coverage with functional tissues while demoting the onset of inflammatory reactions. Findings & Conclusions Here we present a computational tool, based on Monte Carlo simulation, which decouples the contribution of cell proliferation and migration and predicts the cell-separation performance of topographically engineered substrates. Additionally, we propose an optimization procedure to shape the topographically engineered areas of a substrate and obtain maximal cell separation.