Actin-based movement can be reconstituted by using microspheres functionalized with the enzymes N-WASP or ActA, which use the Arp2/3 complex and actin to catalyze the formation of a branched actin filament network that is maintained in rapid turnover by three proteins (capping protein, profilin, and ADF). The particles continuously initiate filament assembly at their surface and are propelled, mimicking bacteria or the leading edge of motile cells. This biomimetic assay offers advantages over approaches based on living cells and cell extracts, because the physical-chemical parameters are under control. The biomimetic motility assay offers the opportunity to test the function of proteins involved in signaling pathways or actin dynamics. It is a powerful tool to understand the physical mechanism of force production and has the potential to support high-throughput screens for drugs, inhibitors of motility, or therapeutic agents in metastatic states in which motility is impaired.