Peptide nucleic acid (PNA) is a chimeric oligonucleotide with nucleotide-derived bases and a peptide backbone. Compared with natural nucleotides, PNA has several advantages, including improved stability and high sequence discrimination during duplex formation. Despite its potential for therapeutic application, analysis technologies have not been generalized mainly due to ambiguous physiochemical properties resembling those of nucleic acids as well as protein. Here, we present a PNA detection method: SDS-PAGE followed by electro-transfer to a Western-blotting membrane and then hybridization with a radio-labeled oligonucleotide probe. This method is useful for evaluating the quality of synthetic PNA and determining its intracellular localization.