Top-down proteomics is an emerging technology based on the analysis of intact proteins using high-resolution mass spectrometry (MS). This chapter gives brief example, showing that top-down proteomics is capable of going beyond matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) for the accurate characterization of bacterial pathogens. In the field of microbiology, several papers have already demonstrated the strength of top-down approaches to study proteins involved in bacterial virulence. This chapter summarizes these studies, as well as future trends. Two distinct analysis strategies exist in top-down proteomics. Choosing which is the most appropriate generally depends on the sample complexity. The first strategy, targeted mode, addresses the analysis of purified proteins or simple mixtures of several proteins and/or proteoforms. The second strategy is discovery mode. In this case, the objective is to perform a large-scale analysis of many proteins present in a “complex” sample, ideally a whole proteome, using a liquid chromatography (LC)-MS/MS strategy.