- The distribution and organization of charges on a protein surface are fundamental properties which affect protein functions and interactions. We demonstrate a new approach for protein surface-charge analysis through modulating protein interactions with chromatic lipid/polydiacetylene (PDA) films. We show that visible and easily quantifiable blue−red transitions, induced on the film surface through electrostatic interactions between the negatively charged PDA and positive soluble species, constitute an effective means for characterizing protein surface charge. Specifically, protein−film interactions can be significantly modulated by complexation between the tested macromolecules and lipid-embedded multivalent calixarene ligands displaying charged residues, making possible protein discrimination based upon the abundance and organization of surface charge. The lipid/PDA film system, in conjunction with the calixarene-derived ligands, facilitates characterization of protein surface charges and identification of anomalous protein electrostatic properties.