Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesising a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor’s resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor’s potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor’s sequence and structure relate to its binding kinetics and function. Using enzyme assays, molecular dynamics simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show the geometry of the binding loop mainly influences the inhibitor’s potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor’s sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin, and two kallikrein-related peptidases, KLK5 and KLK14, that are implicated in various cancers and skin diseases.