Abstract: This paper describes a cell-exclusion patterning method facilitated by a polymeric aqueous two-phase system. The immersion aqueous phase (polyethylene glycol) containing cells rehydrates a dried disk of the denser phase (dextran) on the substrate to form a dextran droplet. With the right properties of the phase-forming polymers, the rehydrating droplet remains immiscible with the immersion phase. Proper formulation of the two-phase system ensures that the interfacial tension between the rehydrating droplet and the surrounding aqueous phase prevents cells from crossing the interface so that cells only adhere to the regions of the substrate around the dextran phase droplet. Washing out the patterning two-phase reagents reveals a cell monolayer containing a well-defined circular gap that serves as the migration niche for cells of the monolayer. Migration of cells into the cell-excluded area is readily visualized and quantified over time. A 96-well plate format of this “gap healing” migration assay demonstrates the ability to detect inhibition of cell migration by known cytoskeleton targeting agents. This straightforward method, which only requires a conventional liquid handler and readily prepared polymer solutions, opens new opportunities for high throughput cell migration assays.