As an alternative material to glass or silicon, microfluidic devices made from a cyclic olefin copolymer (COC) were fabricated. This material is of interest because of the relative ease of fabrication, low costs, and solvent resistance. However, as a result of the strong hydrophobic interactions normally present, COC surfaces are not suitable for protein separations. To reduce the protein adsorption and make COC suitable for protein separations, UV-initiated grafting of polyacrylamide was used to coat the surface of COC devices. The change in surface properties caused by different graft times was studied. The surface hydrophilicity and electroosmotic mobility were characterized by contact angle and electroosmosis measurements. Isoelectric focusing was performed to test protein separations in polyacrylamide-coated COC microchannels. A single protein, carbonic anhydrase, was used to analyze the focusing effects and peak capacities in uncoated and polyacrylamide-coated COC devices. Peak capacities ranging from 75 to 190 were achieved with a polyacrylamide-coated surface. A mixture of two proteins, conalbumin labeled with Alexa Fluor 488 and P-lactoglobulin A labeled with Alexa Fluor 546, was used to test protein separations. Linear and rapid separation of proteins was achieved in the polyacrylamide-coated COC microfluidic device.