Small fluorescent amino acids have emerged as promising building blocks for non-invasive labeling of peptides and proteins. In particular, fluorogenic amino acids are suitable for imaging dynamic events (e.g., interactions with membranes, localization in subcellular organelles) because they provide distinct fluorescence readouts in response to their microenvironments. Our group has reported benzodiazole- based amino acids for solid-phase peptide synthesis (SPPS) and demonstrated their application in fluorescence imaging. One major limitation of the SPPS of fluorogenic peptides is the potential degradation of fluorophores during synthetic steps (e.g., cleavage, removal of protecting groups) as well as their incompatibility with non-protected peptides, such as those obtained by recombinant methods. On the other hand, the incorporation of fluorogenic entities in proteins is largely limited to genetically encoded unnatural amino acids. In this work, we describe a novel late-stage and site-selective functionalization strategy to prepare native-like fluorescent analogues of CPPs and small proteins for realtime imaging of cellular trafficking (Figure ).