Using traditional 2-D photolithographic methods, surface patterns are made on agarose and used to form lipid vesicles with controlled size and layout. Depending on the size and layout of the patterned structures, the lipid bilayer vesicle size can be tuned and placement can be predetermined. Vesicles formed on 2-D patterned surfaces can be harvested for further investigations or can be assayed directly on the patterned surface. Lipid vesicles on the patterned surface are assayed for unilamellarity and protein incorporation, and vesicles are indeed unilamellar as observed from outer leaflet fluorescence quenching. Vesicles successfully incorporate the integral membrane protein α-hemolysin and maintain its membrane transport function.