An increasing number of macrocyclic peptides that cross biological membranes are being reported, suggesting that it might be possible to develop peptides into orally bioavailable therapeutics; however, current understanding of what makes macrocyclic peptides cell permeable is still limited. Here, we synthesized 62 cyclic hexapeptides and characterized their permeability using in vitro assays commonly used to predict in vivo absorption rates, i.e. the Caco-2 and PAMPA assays. We correlated permeability with experimentally measured parameters of peptide conformation obtained using rapid methods based on chromatography and nuclear magnetic resonance spectroscopy. Based on these correlations, we propose a model describing the interplay between peptide permeability, lipophilicity and hydrogen bonding potential. Specifically, peptides with very high permeability have high lipophilicity and few solvent hydrogen bond interactions, whereas peptides with very low permeability have low lipophilicity or many solvent interactions. Our model is supported by molecular dynamics simulations of the cyclic peptides calculated in explicit solvent, providing a structural basis for the observed correlations. This prospective exploration into biomarkers of peptide permeability has the potential to unlock wider opportunities for development of peptides into drugs.