The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.