Currently there are many ion exchangers available for protein purification, but there is little information on how efficiently the ion exchanger surface is used for protein binding. In this work we present the results from a confocal scanning laser microscope study of two anion exchangers, DEAF Toyopearl and Express Ion-Q, after equilibration with protein dye conjugates BSA-BODIPY FL and ovalbumin-Texas Red. The pictures and intensity profiles of DEAF Toyopearl show that both proteins bind preferentially to the surface compared with the core of the spherical ion exchangers. Results on the cylindrical shaped Express Ion-Q with BSA show a similar result but with ovalbumin-Texas Red constant protein binding is observed throughout the adsorbent. Confocal microscopy is demonstrated to be an excellent tool to discriminate between different ion exchangers for the optimisation of protein purification.