AbstractRegulation of gene expression through interaction of proteins with specific DNA sequences is a central issue in functional genomics. Capillary electrophoretic mobility shift assay is an efficient novel method for the investigation of sequence specific protein‐DNA interactions, allowing rapid and sensitive quantification of the complex formation. In this paper, we present a pilot study on capillary zone electrophoretic mobility shift assay (CZEMSA) to investigate the interaction between the transcription factors of HeLa nuclear extract and Sp1‐specific fluorescein‐labeled oligonucleotide, using the unlabeled probe as competitor. The mobility shift assay was accomplished by CZE in coated capillaries without polymeric buffer additives. Specificity of the DNA protein complex formation was verified by competition experiments, as well as by supershift assay with an anti‐Sp1 antibody. The applied electric field strength did not affect the stability of DNA‐protein complex during the electrophoretic analysis, allowing rapid identification and quantification of the protein DNA interaction. A practical application to study the interaction between Oryza sativa MADS‐box transcription factor 4 (OsMADS4) and its consensus sequence is also reported.