SummaryNeutrophil serine proteases (NSPs) exercise tissue‐degrading and microbial‐killing effects. The spectrum of NSP‐mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase‐3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small‐vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life‐threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications.