This protocol describes a method for dynamic patterning cells on a glass coverslip. The glass substrate is first functionalized with photocleavable silane bearing 2-nitrobenzyl group, thereafter a cell-repellent polymer, poly(ethylene glycol) (PEG), is conjugated. Upon absorption of near-UV light, the PEG is cleaved from the surface, changing the surface from non-cell-adhesive to cell-adhesive. The method allows not only for spatially controlling cell attachment on the substrate (conventional patterning), but also inducing cell migration or coculturing heterotypic cells (dynamic patterning). Furthermore, it should be emphasized that the surface is compatible with fluorescence imaging in a high-resolution inverted objective setup as it is composed of a normal glass coverslip functionalized with the thin layers. In this chapter, I describe the procedure for the synthesis of the silane molecule, the preparation of the photoactivatable surface, and its application for dynamic cell patterning.