To validate the use of 4CNW in studying the cell penetration of de novo cyclic peptides, we also substituted 4CNW for an internal tryptophan residue in a recently reported K48 Ub chain-binding cyclic peptide P4, identified using the RaPID system. To assess if 4CNW-P4 was penetrating cells, U2OS cells were incubated with 50 µM peptide for 24h. Cells were then imaged by fluorescence microscopy using the same conditions as described above. As shown in Figure , appreciable intracellular fluorescence was observed suggesting efficient uptake of the cyclic peptide, consistent with data previously reported. Flow cytometry has been widely used to study internalization of fluorescently tagged peptides in cells. Therefore, we wanted to explore the use of 4CNW for monitoring cellular uptake of peptides by flow cytometry. U2OS cells were incubated with 50 µM of 4CNW-P3 or 4CNW-P4 at different time points and the fluorescence of a single cell solution was measured. After 4 h incubation with 4CNW-P4 we could detect a small percentage of positive blue fluorescent cells and this percentage was slightly increased at longer incubation times (Figure , Figure ). Incubation of cells with 4CNW-P3 for 20 minutes was sufficient for initiating the detection of positive cells. These data indicate that flow cytometry is a good technique for monitoring the uptake of peptides containing 4CNW. Label-free assays are the gold standard for determining the cell permeability of cyclic peptide leads. Here, we report the incorporation of ClAc-4CNW and ClAc-AzAla into in vitro translated macrocyclic peptides via initiator reprogramming. In addition, we demonstrate 4CNW and AzAla are good tools for tracking peptide (linear/cyclic) penetration into cells. RaPID selections for de novo cyclic peptides initiated with ClAc-4CNW or ClAc-AzAla will enable the identification of peptide leads that are inherently fluorescent and can be directly assessed for cell permeability by fluorescence microscopy and flow cytometry. 4CNW and AzAla have also recently been used to probe protein-protein binding interactions by PET, FRET and VET. We therefore also envisage that 4CNW/AzAla initiated peptide leads could be used directly in label-free binding assays.