To broaden the range of tools available for proteomic research, we generated a library of 16,368 unique full-length human ORFs that are expressible as N-terminal GST-His(6) fusion proteins. Following expression in yeast, these proteins were then individually purified and used to construct a human proteome microarray. To demonstrate the usefulness of this reagent, we developed a streamlined strategy for the production of monospecific monoclonal antibodies that used immunization with live human cells and microarray-based analysis of antibody specificity as its central components. We showed that microarray-based analysis of antibody specificity can be performed efficiently using a two-dimensional pooling strategy. We also demonstrated that our immunization and selection strategies result in a large fraction of monospecific monoclonal antibodies that are both immunoblot and immunoprecipitation grade. Our data indicate that the pipeline provides a robust platform for the generation of monoclonal antibodies of exceptional specificity.