Herein we report on an approach to use dip-pen nanolithography (DPN) to rapidly prototype nano- and microscale cationic polymer structures that guide the transport of lipid molecules in a fluid membrane. An atomic force microscope (AFM) tip is used to transfer poly(diallyldimethylammonium chloride) (PDAC) to a silica surface that templates the self-assembly of a phospholipid bilayer. Based on AFM and high-resolution optical microscopy data, DPN-generated PDAC structures are optically transparent and composed of up to three molecular layers of polyelectrolyte. We demonstrate that patterns can juxtapose mobile and immobile ligands in a supported lipid bilayer that simultaneously engages the epidermal growth factor receptor (EGFR) and adhesion receptors on the cell surface. In principle, DPN-generated polyelectrolyte structures have important applications in investigating supramolecular protein assemblies in living cells.