Current methodologies for global identification of microbial proteins that elicit host humoral immune responses have several limitations, and are not ideally suited for use in the post-genomic era. Here, we describe a novel application of proteomics, Proteomics-based Expression Library Screening (PELS), to rapidly define microbial immunoproteomes. PELS is broadly applicable to any cultivable, sequenced pathogen eliciting host antibody responses, and hence is ideal for rapidly mining microbial proteomes for targets with diagnostic, prophylactic and therapeutic potential. In this report, we demonstrate “proof-of-principle” by identifying 207 proteins of the Escherichia coli O157:H7 immunome in bovine reservoirs in only three weeks.