We have developed a separation technique for DNAprotein complex based on electrophoretic mobility shift assay (EMSA) by microchip electrophoresis, which we call microchip electrophoretic mobility shift assay (mu EMSA). To evaluate the mu EMSA, we employed recombinant human nuclear factor-?B (rhNF-B) and its consensus double-stranded oligonucleotide (dsOligo) fluorescently labeled with Cy5. We carried out the electrophoretic separation of the consensus dsOligo-rhNF-B complex and the unbound dsOligo in methylcellulose solution and confirmed rapid (similar to 200s) and reliable identification and semi-quantitation of the specific interaction between dsOligo and rhNF-B. The binding specificity of rhNF-?B was confirmed by introducing non-fluorescently labeled consensus oligonucleotide as a competitor. The progression of the binding reaction under various incubation times was monitored, and it was found that the dsOligo and rhNF-B complex formation reached equilibrium (ca. 90% of the dsOligo was bound to rhNF-B) after 5min. Furthermore, without any purification process, even crude NF-?B in nuclear extracts from HeLa cells was specifically detected within 120s by the mu EMSA.