Technical Abstract: Recently, phosphoglucose isomerase with a lysyl aminopeptidase activity (PGI-LysAP) was identified in V. vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: i) a quantitative fluorescent ninhydrin assay for free lysine; ii) matrix-assisted laser desorption/ionization two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and iii) tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-[des-Arg9]-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. The hydrolytic release of free lysine from peptide digests was determined by a rapid, simple, sensitive and quantitative fluorescent ninhydrin assay and was confirmed by MALDI-TOF-TOF. Visualization of peptide hydrolysis was accomplished by tricine gel electrophoresis. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways. Use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies.