A screening method was developed for the fast identification of known pyoverdin-type siderophores produced by fluorescent Pseudomonas spp. It is based on reversed-phase high-performance liquid chromatography interfaced with electrospray ionization mass spectrometry of the Sep-Pak RP-C18 culture supernatant extracts. The siderophores of five bacterial strains were characterized by their molecular masses obtained from their doubly protonated molecular ions [M + 2H]2+ and their UV/visible spectra recorded with a diode-array detector. Additional structural information was gained by skimmer collision-induced dissociation experiments. For all strains new minor siderophores were found. A table of fully or partially identified pyoverdins and related siderophores is provided which will be the basis for screening studies.