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The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4–S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2enhanced the second-order inhibition constant kobs/[I] toward PR3 by more than 10 times (kobs/[I] = 73000 ± 5000 M–1s–1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.