The field of biomaterial engineering is increasingly using high‐throughput approaches to investigate cell–material interactions. Because most material libraries are prepared as chips, immunofluorescence‐based read‐outs are used to uniquely image individual materials. This paper proposes to produce libraries of materials using a well‐based strategy in which each material is physically separated, and thus compatible with standard biochemical assays. In this work, the TopoWellPlate, a novel system to study cell–surface topography interaction in high‐throughput is presented. From a larger library of topographies, 87 uniquely defined bioactive surface topographies are identified, which induce a wide variety of cellular morphologies. Topographically enhanced polystyrene films are fabricated in a multistep cleanroom process and served as base for the TopoWellPlate. Thermal bonding of the films to bottomless 96‐well plates results in a cell culture ready, topographically enhanced, 96‐well plate. The overall metabolic activity of bone marrow‐derived human mesenchymal stem cells is measured to show the functionality of the TopoWellPlate as a screening tool, which showed a 2.5‐fold difference range in metabolic activity per cell. TopoWellPlates of this and other topographical designs can be used to analyze cells using the wealth of standardized molecular assays available and thus disclose the mechanisms of biomaterials‐induced mechanotransduction.