Siderophores are iron chelating molecules that are produced by a variety of microorganisms and have essential functions in building microbial communities. An important group of siderophores are the pyoverdines, a structurally diverse group of chromopeptides with species-specific differences in the peptide sequence and side group structure. Here we describe the development of an LC-MS/MS-based approach to investigate the role of pyoverdines in the interaction of microbial communities. This method utilizes the characteristic fragmentation pattern of all pyoverdines that occurs in the all-ion fragmentation mode and takes advantage of the fact that all pyoverdines are ionized as M2+ ions. In this way, we are able to explore the diversity of pyoverdines and their possible modification.