Peptides, with an intermediate size between small molecules and biologics, are promising probes, therapeutics, and imaging agents. However, peptide agents and therapeutics are often subject to degradation, prompting creative solutions to improve their half-lives and pharmacokinetics. Thioamide substitution, a single-atom O-to-S modification in the peptide backbone, has been shown to improve peptide proteolytic stability while giving the peptide interesting properties. Strategic incorporation of thioamides along with data from past thioamide and sidechain scanning experiments have been leveraged to design peptides for proteolysis studies as well as for imaging and therapeutic purposes. Herein, we demonstrate the design of substrate-based peptide probes, both all-amide and thioamide analogs, for monitoring the proteolytic activity of dipeptidyl-peptidase 4 (DPP-4), which has relevance to diabetes and other metabolic disorders. Secondly, we describe thioamide-based inhibitors of cathepsin L (Cts L), which is a ubiquitously expressed endosomal cysteine protease implicated in many diseases such as cancer, diabetes, and COVID-19; designing specific inhibitors of Cts L against the many highly homologous cathepsin family cysteine proteases has been traditionally challenging. Several thioamide stabilized peptide scaffolds were examined and a good peptidyl substrate was converted into a sub-micromolar inhibitor of Cts L by a single thioamide substitution, with >25-fold specificity against the other cathepsins. This work also showed that the stabilized peptide could inhibit Cts L in human liver carcinoma lysates and in cells. Thirdly, building on previous work in our lab on thioamide-based cancer imaging agents, we successfully designed fluorescently labeled thioamide-based agonists of the neuropeptide Y Y2 receptor – a receptor that is over-expressed in glio- and neuroblastomas. Finally, we report preliminary data regarding thioamides in immunological contexts. Together, this thesis shows the potential of utilizing thioamides as probes, stabilized peptide therapeutics, and reaffirms that this single-atom modification can be added to the toolbox of peptide-based drug design along with imaging applications.