Comprehensive and precise annotations of short protein-coding genes are always a challenging task. Here we propose a new design to facilitate the characterization of previously overlooked short protein-coding genes by integrating a shotgun proteomics method and oligonucleotide array analysis. Using Shigella flexneri as a model, we validate 163 annotated ORFs and 51 hypothetical or putative transcripts at the protein level, and discover four novel short ORFs. This strategy will contribute significantly to comprehensive and accurate genome-wide annotation, and to our understanding of prokaryotic genome structure.