Passive diffusion of small-molecule species across the cell membranes is an essential step in drug delivery since molecules that can easily permeate the plasma membrane have high oral bioavailability. There are several techniques for characterizing the passive transmembrane transport of small molecules; however, there are many instances in which these techniques produce widely varying results. For instance, literature values for the permeabilities of low-molecular-weight carboxylic acids across phosphatidylcholine membranes span three orders of magnitude.We have developed a technique for precisely quantifying lipid membrane permeability to small molecules based on imaging of the concentration field surrounding giant unilamellar lipid vesicle (GUV) membranes. High-speed spinning disk confocal microscopy can be used to image dynamic changes in the concentration field and precisely measure permeabilities as fast as 0.2 cm/s. This technique can be used to measure the transport of both dye-containing species and species that result in a change of fluorescence intensity of a dye molecule. For instance, by using a pH-sensitive fluorophore, the transport of non-fluorescent carboxylic acid molecules can by observed.This technique can be combined with direct imaging of the membrane to simultaneously measure distinct transport rates across different phase-separated regions of a lipid vesicle. We have also imaged transport across GUVs with compositionally asymmetric membranes. These GUVs are formed in a multiphase microfluidic flow system in which the composition of each leaflet of the membrane can be controlled independently. We have measured proton and carboxylic acid transport across membranes with charge asymmetry. Early results show that the permeability of membranes with charge asymmetry depends on the direction in which small molecules and proton species are moving with respect to the membrane.