Here we report a platform for improving the affinity of peptide-based inhibitors of protein-protein interactions using non-canonical amino acids. With this platform—which is inherently selective for high-affinity binders—we realized up to ~100 or ~30-fold gains in affinity for binders to the oncogenic ubiquitin ligase MDM2 or the HIV capsid protein C-terminal domain (C-CA). We demonstrated the utility of the identified compounds as functional PPI inhibitors by rendering them cell permeable via macrocyclization to target MDM2 in cancer cells.