We demonstrate the use of dip-pen nanolithography (DPN) to crystallize proteins on surface-localized functionalized lipid layer arrays. DOPC lipid layers, containing small amounts of biotin-DOPE lipid molecules, were printed on glass substrates and evaluated in vapor diffusion and batch crystallization screening setups, where streptavidin was used as a model protein for crystallization. Independently of the crystallization system used and the geometry of the lipid layers, nucleation of streptavidin crystals occurred specifically on the DPN-printed biotinylated structures. Protein crystallization on lipid array patches is also demonstrated in a microfluidic chip, which opens the way toward high-throughput screening to find suitable nucleation and crystal growth conditions. The results demonstrate the use of DPN in directing and inducing protein crystallization on specific surface locations.