We present the characterization of different polymeric nanoparticles with asymmetrical flow field-flow fractionation (AF-FFF) in different solvents and additional, independent methods such as static and dynamic light scattering (SLS, DLS) in solution and transmission electron microscopy (TEM) and atomic force microscopy (AFM) for the visualization of the nanoparticles on solid substrates. AF-FFF proves to be a powerful technique to determine average sizes of nanoparticles such as multifunctional polyorganosiloxane nanospheres both, in aqueous dispersion and in organic solvents such as toluene. In addition, dye loaded block copolymer vesicles and cylindrical polyelectrolyte type polymacromonomers are successfully analyzed by AF-FFF and the obtained results are compared to the other techniques used.