We present our investigation of using microfluidic devices for rapid protein separation. The devices were made from cyclic olefin copolymers that have high optical clarity and high glass transition temperature. Protein separation was achieved by using isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). A laser-induced fluorescence (LIF) imaging system was developed to detect proteins while they migrated under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers. Dynamic coating of the linear polymers prevented proteins from adsorption and suppressed electroosmotic flows. PAGE was achieved in twenty-nine parallel channels. In addition, we integrated IEF with PAGE in a microfluidic device for two-dimensional protein separation.