Polypeptides are involved in virtually all biological processes due to their ability to specifically interact with other biomolecules. This unique capability, combined with their inherent modular properties, makes polypeptides and their derivatives suitable for engineering and tool development. In this thesis, we first developed a fluorescence polarization assay using peptide substrate to measure the activity of ZDHHC family proteins. We then identified cyano-myracrylamide (CMA), a broad-spectrum DHHC family inhibitor with similar potency to 2-bromopalmitate (2BP). We confirmed that that CMA inhibits DHHC family proteins in cellulo and engages with DHHC family proteins in cells. In contrast to 2BP, CMA has decreased toxicity and avoids inhibition of the S-acylation eraser enzymes. To further explore the potential of using polypeptide for inhibition, we developed an mRNA display platform allowing direct selection of covalent macrocyclic inhibitors using 1,3-dibromoacetone-vinyl sulfone (DBA-VS) as a linker. Testcase selections on TEV protease resulted in potent covalent inhibitors with diverse cyclic structures, among which cTEV6-2, a macrocyclic peptide with a unique C-terminal cyclization, emerged as the most potent covalent inhibitor of TEV protease described to-date, showcasing the application of integrating chemical functionalization and mRNA display in targeted covalent ligand discovery. Finally, we present the Fab-Enabled Split-luciferase Calprotectin Assay (FESCA), a novel quantitative method for calprotectin measurement utilizing two new fragment antigen binding proteins (Fabs) fused with split NanoLuc luciferase fragments, which can be adapted to solution-based or in varied immobilized assay formats and response to elevated calprotectin level in patient samples. FESCA presents a convenient, low-cost, and quantitative method for assessing calprotectin levels in various biological samples, demonstrating the potential of FESCA in improving the diagnosis and monitoring of inflammatory diseases, especially in point-of-care or at-home settings.