We have combined a series of techniques which have previously been used separatelyphosphoramidite synthesis, one-bead-one-compound libraries, fluorescence-activated sorting, and secondary ion mass spectrometry of oligonucleotides -to create a new method which successfully identified the exact number, identity, and sequence position of chemical modifications which result in EGFR affinity-optimised aptamers. By comparison with the parent MinE07 aptamer and uniformly modified controls, we have shown that performing this selection makes a valuable difference to the aptamer affinity for EGFR. Since our method does not use enzymatic amplification, all the chemical information about the modifications is retained throughout the selection and sequencing workflow, which means that chemical optimization can now be performed orthogonally to the aptamer nucleobase sequence. In doing so, the aptamer can be made more fully complementary to the chemical features of the target, bringing them closer to the versatility of antibodies. This has the potential to convert known, but poorly performing aptamers for important targets into systems which could now be applied in drug discovery.