- Purpose. The aims of this study are to develop a rapid colorimetric assay for evaluating membrane interactions and penetration through lipid barriers and to create a platform, amenable to highthroughput screening formats, for predicting the extent of penetration of pharmaceutical compounds through lipid layers. Methods. The colorimetric platform comprises vesicles of phospholipids and the chromatic lipidY mimetic polymer polydiacetylene. The polymer undergoes visible, concentration-dependent blueYred transformations induced through interactions of the vesicles with the molecules examined. Results. We observe rapid colorimetric transitions induced by addition of pharmaceutical compounds to the chromatic vesicle solutions. We find that the concentration ranges for which the color transitions are induced in the lipid/polymer vesicles are correlated with the degree of lipid interactions and bilayer penetration of the tested compounds. The colorimetric platform could distinguish between three primary types of membrane-permeation profiles: bilayer-surface attachment, membrane penetration, and absence of lipid interactions. Application of complementary bioanalytical techniques corroborated the interpretation of the colorimetric data. Different pharmaceutical compounds were tested by the new assay. The results indicated clearly distinct membrane interaction profiles for molecules expected by conventional methods to have similar membrane-insertion properties (i.e., close log D/log P values). In addition, the new colorimetric assay pointed to similar membrane activities for molecules having highly divergent log Ds. Conclusions. The colorimetric assay facilitates Bcolor coding’’ that could distinguish among different membrane permeation profiles. The data point to the usefulness of the platform for characterization of drug compound interactions with lipid assemblies. The new colorimetric technology constitutes a generic, extremely fast, and easily applicable approach for predicting and screening interactions of pharmaceutical compounds with lipid barriers.