Proteins that interact with nucleic acids are central to numerous cellular processes and their continuing characterisation represents one of the foremost challenges in the post-genomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce, and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore, it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.