Patterning multiple antibodies (each to a different antigen) on a single substrate is an important step in the development of multi-analyte biosensors. The envisioned device requires patterning antibodies onto a substrate in discrete pixels, with adjacent pixels containing antibodies against different antigens. In this paper, a new method for forming patterns of different antibodies on a surface is described. The method utilizes hydrophobic interactions to immobilize antibodies on a polystyrene surface. Prior to incubation in the antibody, the substrate is coated with bovine serum albumin (BSA) to prevent nonspecific adsorption. The BSA is then selectively removed from the region where antibody adsorption is desired. Results will be presented demonstrating several methods of selective BSA removal, including ion-beam sputtering and mechanical scribing. Ion beam sputtering has been used to form a rudimentary, millimeter-scale pattern of two antibodies on a polystyrene surface. X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were used to detect the spatial distribution of 10 nm gold labeled antigen on the surface. Mechanical scribing has been used to form a 0.1 mm-scale pattern of two antibodies on a surface. Fluorescence Microscopy was used to visualize the FITC labeled antigen on the surface. Finally, results will be presented showing the use of the AFM as a lithographic tool for antibody patterning. The NSWC, Carderock Division In-House Laboratory Independent Research Program provided funding support for this work.