Proteases are the biological catalysts for a simple yet crucial reaction in living organisms, the hydrolysis of amide bonds. Beyond the broad-spectrum degradation of proteins, proteases also play key roles in sophisticated physiological processes, and their dysfunction and misregulation are implicated in a wide range of human diseases. Modified protease substrates are developed as fluorescent probes to monitor protease activity, and as peptide therapeutics to treat protease-regulated diseases. However, current protease substrate modifications are often limited by their relatively large sizes, resulting in measurements of disrupted protease activity or in compromised therapeutic activity. Here, we showed that the thioamide, a single-atom substitution in an amide bond, can be introduced into protease substrates to develop non-perturbing protease activity probes, or to stabilize peptide therapeutics against proteolysis without reducing their physiological activity, depending on the modification position. When placed away from the scissile bond of substrates, thioamides can be paired with fluorophores to create fluorescent turn-on probes to monitor protease activity in real-time under various settings. We have demonstrated that this strategy can be used to study protease kinetics, create highly specific probes for the target protease, and monitor protease activity in cell lysates. When placed near the scissile bond of substrates, thioamides can extend the half-life of peptide therapeutics against proteolysis by over 1000-fold while maintaining identical bioactivity. Thioamide-modified anti-diabetic peptides glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP) show prolonged in vitro half-life and identical potency to activate cell surface receptors. The best performing GLP-1 thioamide analog was tested in an oral glucose tolerance test (OGTT) in rats, and dose-dependent glycemic control superior to that of GLP-1 was observed. In addition, the positional effects of thioamide modification on protease activity was systematically investigated to provide guidance of where to place thioamides. Such studies begin to establish rules for how thioamides can be employed in sensor and in peptide stabilization applications.