Based on the enzyme specificity of matriptase, a type II transmembrane serine protease (TTSP) overexpressed in epithelial tumors, we screened a cDNA library expressing variants of the protease inhibitor eglin c in order to identify potent matriptase inhibitors. The most potent of these, R 1 K 4 ′‐eglin, which had the wild‐type Pro 45 (P1 position) and Tyr 49 (P4′ position) residues replaced with Arg and Lys, respectively, led to the production of a selective, high affinity ( K i of 4 nM) and proteolytically stable inhibitor of matriptase. Screening for eglin c variants could yield specific, potent and stable inhibitors to matriptase and to other members of the TTSP family.