Electric field gradient focusing (EFGF) is a separation technique that uses an electric field gradient and an opposing hydrodynamic flow to separate and concentrate charged analytes. This work describes miniaturized EFGF devices that are used for protein analysis. These devices employ a unique ionically conductive polymer that enables the required electric field gradient to be established. This polymer has good protein compatibility and allows the transport of small buffer ions while retaining large analytes such as proteins. With the use of an EFGF device, green fluorescent protein was concentrated 10 000-fold and the separation of a protein mixture was demonstrated. The development of these ionically conductive polymer-based devices represents a step toward making EFGF a useful analytical tool for proteomics investigations.