Liquid chromatography coupled with mass spectrometry (LC-MS) is the method of choice for analysis of covalent modification of DNA. DNA adductomics is an extension of this approach allowing for the screening for both known and unknown DNA adducts. In the research reported here, a new high-resolution/accurate mass MSn methodology has been developed representing an important advance for the investigation of in vivo biological samples and for the assessment of DNA damage from various human exposures. The methodology was tested and optimized using a mixture of 18 DNA adducts representing a range of biologically relevant modifications on all four bases and using DNA from liver tissue of mice exposed to the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK). In the latter experiment, previously characterized adducts, both expected and unexpected, were observed.