A microfluidic device that performs in space sample fractionation, collection, and preconcentration for proteomics is described. Effluents from a 2.75 mm long fractionation channel, focused via sheath flow, were sequentially delivered into an array of 36-collection channels containing monolithic polymer beds for SPE. Optimum conditions for the device design, and simultaneous photolytic fabrication of 36 monolithic columns in the 36 channels, as well as for their proper performance in electrokinetic sample fractionation and collection are described. A hydrophobic butyl methacrylate-based monolithic porous polymer was copolymerized with an ionizable monomer, acryloamido-methyl-propane sulfonate, to form a polymer monolith for SPE that also sustains cathodic electroosmotic flow. The SPE bed was made deep enough to greatly reduce the linear flow rate within the bed, in order to compensate for the lower electroosmotic mobility of the cationically charged SPE bed relative to the glass walled device. Under these conditions, electrokinetic fractionation of a protein sample resulted in tightly focused sample zones delivered into each of the 36-channel polymer beds with no observed crosscontamination. Monolithic columns showed reproducible performance with preconcentration factor of 30 for 2 min loading time. The ability to fractionate, collect, and preconcentrate samples on a microfluidic platform will be especially useful for automated or continuous operation of these devices in proteomics research.