A low-sized, uniform and polymer-based high-performance liquid chromatography (HPLC) packing material capable of making reversed-phase separation of proteins with high resolution and with high column efficiency was developed. By a multi stage-swelling and polymerization protocol, 5 mu m-uniform-porous poly(styreneco-divinylbenzene) particles with relatively larger pores particularly suitable for protein separation were synthesized by starting from a low-sized seed latex with high average molecular weight and by using a diluent phase comprised of dibutylphthalate and toluene. By the use of synthesized beads as packing material in HPLC, highresolution liquid chromatograms were obtained in the gradient separation of selected proteins (i.e., ribonuclease-A, lysozyme, cytochrome C, and albumin). In the chromatographic runs, the flow rate of the mobile phase was increased fourfold by preserving the resolution power of the column material under gradient conditions. The theoretical plate numbers (TPN) up to 12.500 plates/m were observed by using cytochrome C as the analyte. TPN values determined by the proteins were significantly higher relative to the similar uniform packing materials larger in size (i.e., 7.5 - 10 mu m) obtained by different polymerization methods.