Many biological processes are governed by protein-protein interactions (PPIs). Development of small molecules modulating these interactions can be difficult due to large and shallow interaction interfaces. Biologics, such as antibodies, are ideal for targeting PPIs, but they lack cell-permeability. Macrocycles occupy an intermediate space between small molecules and biologics, having sufficient size and functionality to interact specifically and with high affinity with PPI surfaces. This is well precedented by natural products, many of which are marketed drugs. Natural products, however, can be difficult to identify and analog, so we have been using mRNA display as a means to identify and optimize peptide-based macrocycles. While the benefits of peptide-based macrocycles are significant, they also have challenges as drug leads, such as low oral bioavailability and poor membrane permeability. We have been applying multiple methods, such as NMR, chromatographic techniques, computational tools and biological assays, to explore and understand the “rules” governing macrocycle permeability. Ultimately, we want to generate macrocycle libraries through mRNA display, composed of both natural and non-natural amino acids, that are biased towards cell-permeable scaffolds.