Large unilamellar vesicles of POPC were prepared by hydrating a lipid film using an internal solution, followed by 9 freeze-thaw cycles and extrusion 25 times through a 200 nm polycarbonate membrane. Finally, the vesicles were subject to dialysis or Sephadex size-exclusion chromatography using the required external solution to remove unencapsulated chloride ions (ISE assay) or the fluorescence pH indicator 8-hydroxypyrene-1,3,6-trisulfonate (HPTS assay) to obtain vesicle stock suspensions for membrane transport studies.