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This study presents a novel and easily applicable approach to recruit sulfhydryl-containing biomolecules to membranes by using a palmitic acid which is functionalized with a maleimide group. Notably, this strategy can also be employed with preformed (biological) membranes. The applicability of the assay is demonstrated by characterizing the binding of a Rhodamine-labeled peptide to lipid and cellular membranes using methods of fluorescence spectroscopy, lifetime measurement, and microscopy. Our approach offers new possibilities for preparing biologically active liposomes and manipulating living cells.