In recent years cyclic peptides have emerged as promising drug candidates that can combine therapeutic benefits from both small molecules and biologics. They show excellent selectivity and tight binding to their targets, and their ability to interact with shallow surfaces makes them suitable for tackling challenging surfaces such as those involved in Protein-Protein Interactions. High affinity de novo cyclic peptides can be efficiently identified from enormous encoded peptide libraries using e.g. Phage or mRNA display platforms (10 8 -10 14 members). In particular the Random nonstandard Peptides Integrated Discovery (RaPID) allows the identification of cyclic peptides containing a wide range of unnatural amino acids. One major challenge for the application of peptides to intracellular targets, however, is their low inherent cell permeability. Straightforward assays are therefore required to assess the permeability of promising leads, with fluorescence imaging being a popular approach. Typically, peptide leads are appended with a fluorophore (e.g. FITC, TAMRA, Rhodamine) to assess cell permeability. Whilst widely used, these strategies use the labelled peptide as a proxy for the untagged version of the molecule. This can result in misleading findings where the fluorophore changes the physicochemical properties of the parent peptide and influences their ability to penetrate and localize in cells. Use of minimally perturbing dyes, or label-free assays are therefore the gold standard for determining the cell permeability of cyclic peptide leads.