A new method was developed to purify membrane-bound species within a supported lipid bilayer (SLB) environment. SLBs consisting of phosphatidylcholine lipids and cholesterol were employed as the separation matrix. Cholesterol was used to reduce the diffusion of lipids within the bilayer and, therefore, substantially reduce mixing of the dye-conjugated lipids to be separated. These molecules were introduced into an SLB adjacent to the separation SLB, and electrophoresis was employed to move these species through it. The method was powerful enough to completely resolve two isomers of Texas Red DHPE from each other. Moreover, these isomers could be separated from a BODIPY-conjugated lipid as well. Such procedures could be extended to the purification of peripheral and transmembrane proteins.