We report a novel approach to quantitatively determine complete size distributions of surface-bound objects using fluorescence microscopy.Wemeasure the integrated intensity of single particles and relate it to their size by taking into account the object geometry and the illumination profile of the microscope, here a confocal laser scanning microscope. Polydisperse (as well as monodisperse) size distributions containing objects both below and above the optical resolution of the microscope are recorded and analyzed. The data is collected online within minutes, which allows the user to correlate the size of an object with the response from any given fluorescence-based biochemical assay. We measured the mean diameter of extruded fluorescently labeled lipid vesicles using the proposed method, dynamic light scattering, and cryogenic transmission electron microscopy. The three techniques were in excellent agreement, measuring the same values within 7–9%. Furthermore we demonstrated here, for the first time that we know of, the ability to determine the full size distribution of polydisperse samples of nonextruded lipid vesicles. Knowledge of the vesicle size distribution before and after extrusion allowed us to propose an empirical model to account for the effect of extrusion on the complete size distribution of vesicle samples.