Neutrophils play an essential role in the innate immune system, where they defend against invading pathogens by engulfing and degrading various microorganisms. Neutrophil serine proteases (NSPs) are expressed by neutrophils and contribute to killing microorganisms, stimulating inflammation and remodeling the extracellular matrix. One of the four NSPs is proteinase 3 (PR3), which participates in each of the three processes listed above. However, an imbalance between PR3 activity and its inhibition has been identified in several diseases that feature chronic inflammation in the lungs, such as chronic obstructive pulmonary disease (COPD). Therefore, PR3 is becoming increasingly recognised as a therapeutic target in diseases related to chronic inflammation and selective inhibitors for PR3 have the potential to serve as therapeutic leads. In this study, PR3 inhibitors were designed based on sunflower trypsin inhibitor-1 (SFTI-1), a 14-amino acid cyclic peptide that was discovered in sunflower seeds. According to previous substrate screening results, amino acids preferred by PR3 at each of the major binding sites (P1, P2, P2ʹ, P4 and P5ʹ) were selected to substitute into the SFTI-scaffold. Positions were screened one by one to progressively optimize the inhibitor’s sequence, which led to the design of a potent PR3 inhibitor, SFTI-Nva* (sequence: GBipCYNvaSYPPICNPN), with a Ki value against PR3 of 6.1±0.6 nM.