Staphylococcus aureus is a notorious human pathogen that relies on an array of virulence factors to engender infection and evade the host-immune system. Among these are the secreted proteases, which promote pathogenesis by degrading host proteins and modulating host-defenses. Human neutrophils play a pivotal role in these defenses, acting as the first responders against invading bacteria. While many S. aureus effectors of virulence have been shown to target leukocytes, there is limited knowledge on how the extracellular proteases modulate neutrophil fate. Typically, protease substrates have been identified in isolated settings using one at a time approaches; with neutrophil targets few and far between. Herein, we have developed a novel N-terminomic methodology termed TAGS-CR that can facilitate global substrate characterization in streamlined manner. We thus present the application of TAGS-CR to unravelling the human neutrophil pathodegradome of the S. aureus V8 protease. In so doing, we captured ~350 V8 targets, revealing critical insight into how this virulence factor can modulate neutrophil functionality on various levels relevant to S. aureus disease progression. We recorded cleavage of proteins necessary for neutrophil adhesion and migration, a fundamental process necessary for pathogen clearance. Furthermore, we highlight V8 cleavage of proteins involved in important neutrophil defense tactics, such as degranulation and reactive oxygen species production. This protease may also facilitate bacterial dissemination via the intentional activation of neutrophil apoptosis. Collectively, this work deepens our understanding of host-pathogen interaction and begins to unravel how S. aureus proteases can induce immune dysregulation through the targeting of leukocytes.