A method for accurate mass measurement of oligonucleotides up to a DNA 35-mer based on matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is described. In this method, a time-lag focusing time-of-flight mass spectrometer is used to achieve high mass resolution to resolve adduct ions that often complicate the mass analysis of oligonucleotides. Mass resolutions between 1170 and 1300 (full width at half maximum) for a 17-mer, 23-mer, and 35-mer are obtained using a 1 m linear time-of-flight instrument with a total sample loading of less than 10 pmol. The effects of sample preparation, type of calibrant and matrix used on the accuracy of mass measurement, based on external calibration, are discussed. A sample preparation protocol that forms a thin film of matrix and sample crystals on a MALDI probe is described. It is shown that mass measurement error less than 100 ppm with reproducibility better than +/-60 ppm can be obtained with either proteins or DNA fragments as external calibrants. Accurate mass measurement for a mixture of DNA fragments is also illustrated.