This paper investigates three different approaches to patterning proteins within ultrathin resist layers formed from self-assembled monolayers using scanning probe lithography (SPL) at the submicrometer length scale. The first approach uses a “nanografting” method to pattern a reactive carboxylic acid terminated thiol into a resist composed of a methyl-terminated monolayer. Rabbit IgG antigen is bound to the patterned region, and an immunoassay utilizing direct readout of the topographic change resulting from specific binding of anti-rabbit IgG antibody is performed using scanning force microscopy. To address issues related to nonspecific protein adsorption, the other two approaches investigated the patterned removal of glycol-terminated monolayers by mechanically “scraping” patterns at high tip−sample forces by SPL. Protein attachment to the scraped regions was achieved either through the chemisorption of a disulfide coupling agent or by the direct adsorption of Fab‘-SH antibody fragments. Results obtained from all approaches are presented and compared, and the strengths and weaknesses of each toward fabricating high-density, multiple protein arrays are discussed.