Abstract: Methods for determining the sequence of nucleic acids by cleaving the nucleic acid unilaterally from a first end with an exonuclease activity to sequentially release individual nucleotides, identifying each of the sequentially release nucleotides by mass spectrometry, and determining the sequence of the nucleic acid from the identified nucleotides are disclosed. The method is amenable to multiplexing for simulataneously determining more than one nuleic acid sequence.