Vesicle formation by a staggered herringbone microfluidic mixer was investigated in comparison to a sonication-extrusion method. Experiments focused on the incorporation efficiency of lipid components, on dye entrapment efficiency, and on the barrier properties of the vesicle bilayers produced. The microfluidic method produces vesicles largely under the control of thermodynamic factors. As a result, the molecular parameters of the lipids(chain length, chain volume, head group area) directly control vesicle diameter. A hydrophobic branched chain sulfonate lipid was incorporated by microfluidic mixing but not by sonication-extrusion. The vesicles produced by microfluidic mixing can be used to study ion transport by known ionophores and appear to have directly comparable barrier properties to those produced by sonication-extrusion. Vesicles containing the branched chain sulfonate are highly permeable. The microfluidic mixing method produces predominantly unilamellar vesicles.