Targeted delivery of peptides and proteins into intracellular compartments remains a major challenge in macromolecule therapeutics because of the complexity and diversity of cellular uptake mechanisms. For cell-penetrating peptides (CPPs), different models of direct translocation have been proposed, which include carpet-like membrane disruption or the formation of transient pores or inverted micelles. CPPs can enter cells via endocytosis, an energy-dependent process that involves membrane binding, endosomal uptake and endosomal release. On the other hand, small proteins (e.g., chemokines) can enter cells via receptor-mediated endocytosis, a process that requires binding to membrane receptors for active internalization. In order to enhance our understanding of cell internalization pathways and trafficking of peptides and small proteins in live cells, fluorescence microscopy studies require labeling strategies that preserve their main physicochemical features while incorporating suitable reporters for real-time imaging. The derivatization of peptidic structures -including CPPs and small proteins-with commercial fluorophores has been used to investigate cell uptake and trafficking. Many fluorophores are based on relatively hydrophobic or charged structures that can alter the secondary structure and internalization mechanisms of native sequences, thus leading to inaccurate observations when studying macromolecule internalization. Other assays consider chemical tags that can be detected by interaction with secondary components (e.g., HaloTag), which have been described to quantify the cytosolic delivery of CPPs. However, this approach requires genetically-modified cells lines and cannot provide mechanistic insights on the internalization routes of peptides or proteins.