Cellular membranes employ a variety of strategies for controlling the flow of small molecules into the cytoplasmic space, including incorporation of sterols for modulation of permeability and maintenance of lipid asymmetry to provide both sides of the membrane with differing biophysical properties. The specific case of cholesterol asymmetry, especially, is known to have profound effects in neurological cellular systems. Synthetic membrane models that can readily determine valuable physical parameters, such as water transport rates, for sterol-containing membranes of defined lipid composition remain in demand. We report the use of the droplet interface bilayer (DIB), composed of adherent aqueous droplets surrounded by a lipid monolayer and immersed in a hydrophobic medium, for measurement of water permeability across the membrane, with rapid visualization and ease of experimental setup. We studied droplet bilayer membranes composed of the prototypical synthetic membrane lipid (i.e., the archaeal lipid DPhPC) as well as of symmetric and asymmetric DIBs formed by DPhPC and sodium cholesterol sulfate (S-Chol). The presence of S-Chol in DPhPC in symmetric DIB reduced the passive water permeability rate (P-f) at all concentrations and increased the activation energy (E-a) to 17-18 kcal/mol. When only one side of the DIB contains S-Chol (asymmetric DIB), an E-a of 14-15 kcal/mol was obtained, a value intermediate that of pure lipid and symmetrical DIB containing lipid and S-Chol. Our data are consistent with a capability for regulation of water transport by one leaflet independent of the other. The engineering of our various systems is believed to have implications for garnering detailed knowledge regarding the transport of small moieties across bilayers in a wide variety of lipid systems.