In recent years, the demand for quantitative measurement of DNA has been increased in the field beyond basic biology, such as food analysis and clinical diagnosis. Currently, real-time PCR, DNA microarray and UV absorption are utilized for the quantitation, however, the accurate quantitative measurement for DNA has not been established, because of the lack of well characterized reference materials. We have developed an accurate quantitative analysis of DNA using isotope-dilution mass spectrometry (ID-MS) for the development of DNA reference materials. In this study, 20-mer oligo DNA was used for the analyte, which was spiked with isotopically labeled nucleotides, and digested enzymatically. The resulted nucleotides and nucleosides mixtures were measured by LC-MS. The measured concentration of the analyte oligonucleotide was compared with other quantitation methods. The accuracy of this method has also been verified by the inter-laboratory comparison based on the CIPM/CCQM pilot study P54.1.