Peptides offer distinct advantages as a protein targeting modality for mechanistic probe and drug development. Given their large surface area and the potential for highly tailored design, peptide ligands can have exquisite target-binding affinities and specificities. To counteract conformational and metabolic instabilities, many peptide stabilization strategies have been deployed, in some cases leading to cell-permeable bioactive compounds. A noteworthy peptide stabilization chemistry is ring-closing olefin metathesis, used in the formation of stapled peptides, constrained alpha-helical peptides that serve as protein-protein interaction inhibitors. Herein we report first-in-class cell-active stapled peptide inhibitors of RAB25, a recalcitrant protein target implicated in the pathogenesis of a variety of cancers. This work exemplifies the potential of stabilized peptides to engage challenging targets in relevant biological contexts, however the current repertoire of peptide stabilization chemistries have several limitations, including: the requirement of exogeneous reagents or harsh conditions often incompatible with unprotected peptide, full length proteins, or aqueous conditions; a narrow scope of applicable folds and resulting stabilized structures; limited diversity in stabilizing linker structure, which rigidifies peptide conformation and can itself impact target-binding. To begin addressing these shortcomings, we applied the Diels-Alder reaction as a bioorthogonal chemistry for peptide cyclization. Our studies confirm its suitability for both on-resin and in-solution peptide cyclization, in organic and aqueous solutions respectively. Cyclization kinetics are rapid and high-yielding across a range of diene and dienophile functional groups and peptide folds, predominantly resulting in cycloadducts with endo stereochemistry as ascertained by NMR and X-ray crystallographic studies. Further, Diels-Alder cyclized peptides display enhanced bioactivity, with cycloadduct composition and geometry having differential impacts on target-binding, confirmed by the observation of substantial cycloadduct-protein contacts in a crystal structure of an SRC2-derived Diels-Alder cyclized peptide bound to its target estrogen receptor alpha. Additionally, Diels-Alder peptide cyclization is shown to be compatible with both ring-closing olefin metathesis and disulfide formation, suggesting the broad applicability of this chemistry alongside other stabilization chemistries. Separate work reports on the design and screening of peptide inhibitors targeting a highly conserved coronavirus methyltransferase complex, nsp16/nsp10, central to the infectivity of SARS-CoV-2, the cause of the ongoing COVID-19 global pandemic. Finally, we present a robust, multi-readout cell penetration, compound stability, and membrane disruption assay for peptides and other synthetic biologics. This allows for conclusive reporting of key pharmacologic properties of these promising protein-targeting modalities. Taken together, these works serve to expand the application, function, and study of synthetic biologics and related compounds for probing biology and treating disease.