We herein report a method for identification of the location and type of chemical modifications to an existing aptamer to improve its affinity -selection of optimised ligands by fluorescence-activated bead sorting (SOLFABS). The method achieves this by combining high-precision automated flow cytometrybased selection with enzyme-free sequence readout, retaining all the information concerning modification locations. This will further boost the future success of aptamers as therapeutics, targeting agents, and in diagnostics.