The emergence of antibiotic-resistant bacteria has intensified the need for novel therapeutic strategies targeting bacterial virulence rather than growth or survival. Bacterial virulence involves complex processes that enable pathogens to invade and survive within host cells. Chemical biology has become a powerful tool for dissecting these virulence mechanisms at the molecular level. This review highlights key chemical biology approaches for studying bacterial virulence, focusing on four areas: 1) regulation of virulence, where chemoproteomics has identified small molecule-protein interactions that modulate virulence gene expression
2) identification of virulence proteins, using techniques like unnatural amino acid incorporation and activity-based protein profiling (ABPP) to uncover proteins involved in infection
3) post-translational modifications of host proteins, where chemical probes have revealed how bacterial effectors alter host cell processes
and 4) effector-host protein interactions, with methods such as bifunctional unnatural amino acid incorporation facilitating the discovery of key host targets manipulated by bacterial effectors. Collectively, these chemical tools are providing new insights into pathogen-host interactions, offering potential therapeutic avenues that aim to disarm pathogens and combat antibiotic resistance.