Here we present a method to simultaneously characterize and/or optimize both the binding loop towards the protease and a cysteine-stabilized scaffold. The small peptidic sunflower trypsin inhibitor (SFTI-1) was chosen as a model system for these experiments. The inhibitor was investigated for positional specificity against trypsin, elastase and proteinase K using complete substitutional analyses based on cellulose-bound peptide spot synthesis. Inhibitor variants optimized for elastase or proteinase K inhibition by several rounds of substitutional analyses exhibit K(i) values in the micromolar range and high specificity for the corresponding protease. The results of this easy-to-perform assay can be used to design an improved peptide library using classical methods.