Plant seeds are a rich source of protease inhibitors that contribute to protecting seeds from digestion and aid seed dispersal. One of the smallest seed-derived protease inhibitors is a 14-amino acid cyclic peptide found in sunflower seeds, sunflower trypsin inhibitor-1 (SFTI-1), which shows strong activity against trypsin from humans and other species, with inhibition constants in the low picomolar range. Trypsin is a prototypic member of a large family of serine proteases that have evolved to carry out a diverse spectrum of biological functions that are prominent in homeostasis and disease, yet each enzyme shares a common structural fold. Our work on engineered SFTI-based inhibitors parallels the structural conservation‒functional diversity seen in trypsin-like proteases in that our design strategy uses SFTI-1 as a scaffold that has been pre-optimized to target the serine protease active site, and can be equipped with activity against new proteases by customizing its sequence. Using different approaches to guide sequence optimization of the SFTI scaffold, we have developed a series of engineered inhibitors for separate proteases targets, including proteases where the naturally occurring scaffold lacks activity. Several of these inhibitor variants have now been used in chemical biology studies to provide new insight into the biological functions of different proteases.