This study investigates how the S1 and S3 site of trypsin can be challenged with cationic amino acid analogues to yield active antimicrobial peptides with stability toward tryptic degradation. It is shown that unnatural analogues can be incorporated to generate stable peptides with maintained bioactivity to allow for a potential oral uptake. Selected peptides were studied using isothermal calorimetry and computational methods. Both stable and unstable peptides were found to bind stoichiometrically to trypsin with dissociation constants ranging 2-60 μM, suggesting several different binding modes. The stability of selected peptides was analyzed in whole organ extracts and the incorporation of homoarginine and 2-amino-(3-guanidino)propanoic acid resulted in a 14- and 50-fold increase in duodenal stability. In addition, a 40- and 70-fold increase in stomach stability is also reported. Overall, these results illustrate how the incorporation of cationic side chains can be employed to generate bioactive peptides with significant systemic stability.