The fate of a dodecathymidine prodrug in cell extract was monitored by MALDI-TOF MS. This technique allows a facile identification and a relative quantification of metabolites produced. We showed that the relative peak intensities were similar to the relative metabolite proportions that permitted the determination of their half-lives. We found a good fit between the calculated kinetics curves and the experimental points. The oligonucleotide prodrug was fully metabolized to yield the dodecathymidine phosphorothioate likely through a carboxyesterase mediated mechanism.