Amphiphilic molecules such as surfactants, lipids, and block copolymers can be assembled into bilayers and form vesicles. Fluorescent membrane labeling methods require the use of dye molecules that can be inserted into the bilayers at different stages of synthesis. To our knowledge, there is no generalized method for labeling preformed vesicles. Herein, we develop a versatile protocol that is suitable to both surfactant and lipid preformed vesicles and requires no separation or purification steps. On the basis of the lipophilic carbocyanine green dye PKH67, the methodology is assessed on zwitterionic phosphatidylcholine vesicles. To demonstrate its versatility, it is applied to dispersions of anionic or cationic vesicles, such as a drug administrated to premature infants with respiratory distress syndrome, or a vesicle formulation used as a fabric softener for home care applications. By means of fluorescence microscopy, we then visualize the interaction mechanisms of nanoparticles crossing live cell membranes and of surfactants adsorbed on a cotton fabric. These results highlight the advantages of a membrane labeling technique that is simple and applicable to a large number of soft matter systems.