Bacterial small ORF (sORF)-encoded polypeptides (SEPs) contribute significantly to both the basic physiology and infection biology of bacteria. Yet, because of the challenges their small sizes and biochemical peculiarities pose for standard protein detection methods like mass spectrometry and immunoblotting, the bacterial small protein world remains largely unexplored. Starting from a set of putative SEPs inferred from riboproteogenomics data, this project aims to investigate the challenging nature of small protein detection and advance toward an optimized workflow for the full annotation of bacterial SEPs. Using Salmonella Typhimurium as a model pathogen, we here set off to functionally characterize the protein products of these fascinating novel genomic elements. Following the genome-wide discovery of putative novel sORFs, SEP expression will be validated followed by studies on SEP stability, localization, and interactions using state-of-the-art innovative omics strategies to further assign biological functions. Such an integrative and systematic SEP-centric effort will provide unprecedented insights into bacterial SEP biology and holds the potential to highlight candidates for antimicrobial treatments and diagnostic tools.