A near-infrared fluorescent (NIRF) substrate-based probe (SBP) was conceived to monitor secreted human proteinase 3 (hPR3) activity. This probe, called pro3-SBP, is shaped by a fused peptide hairpin loop structure, which associates a hPR3 recognition domain (Val-Ala-Asp-Nva-Ala-Asp-Tyr-Gln, where Nva is norvaline) and an electrostatic zipper (consisting of complementary polyanionic (d-Glu)5 and polycationic (d-Arg)5 sequences) in close vicinity of the N- and C-terminal FRET couple (fluorescent donor, sulfoCy5.5; dark quencher, QSY21). Besides its subsequent stability, no intermolecular fluorescence quenching was detected following its complete hydrolysis by hPR3, advocating that pro3-SBP could further afford unbiased imaging. Pro3-SBP was specifically hydrolyzed by hPR3 (kcat/Km= 440 000 ± 5500 M–1·s–1) and displayed a sensitive detection threshold for hPR3 (subnanomolar concentration range), while neutrophil elastase showed a weaker potency. Conversely, pro3-SBP was not cleaved by cathepsin G. Pro3-SBP was successfully hydrolyzed by conditioned media of activated human neutrophils but not by quiescent neutrophils. Moreover, unlike unstimulated neutrophils, a strong NIRF signal was specifically detected by confocal microscopy following neutrophil ionomycin-induced degranulation. Fluorescence release was abolished in the presence of a selective hPR3 inhibitor, indicating that pro3-SBP is selectively cleaved by extracellular hPR3. Taken together, the present data support that pro3-SBP could be a convenient tool, allowing straightforward monitoring of human neutrophil activation.