AbstractWe developed a method for separating liposomes by size and determining their average diameters. Liposomes with different average diameters were separated on a monolithic silica capillary column, and the size of the liposomes corresponding to each peak was determined online with a dynamic light scattering detector coupled to the capillary liquid chromatography system. The calculated diameters for the separated liposomes were similar to the diameter values measured in batch mode. We demonstrate that this combination of a monolithic capillary column and light scattering detection could be used for size separation of liposomes and could provide more details about average diameters than batch‐mode analysis.