The ability to manipulate single protein molecules on a surface is useful for interfacing biology with many types of devices in optics, catalysis, bioengineering, and biosensing. Control of distance, orientation, and activity at the single molecule level will allow for the production of on-chip devices with increased biological activity. Cost effective methodologies for single molecule protein patterning with tunable pattern density and scalable coverage area remain a challenge. Herein, Hole Mask Colloidal Lithography is presented as a bench-top colloidal lithography technique that enables a glass coverslip to be patterned with functional streptavidin protein onto patches from 15-200 nm in diameter with variable pitch. Atomic force microscopy (AFM) was used to characterize the size of the patterned features on the glass surface. Additionally, single-molecule fluorescence microscopy was used to demonstrate the tunable pattern density, measure binding controls, and confirm patterned single molecules of functional streptavidin.