Alternative approaches complementing the existing technologies for analysis of nucleic acids and their assemblies are necessary to take on the new challenges posed by the postgenomic era. The versatility of MS in biopolymer analysis and its ability to reach beyond sequence information are the basis of ever expanding applications aimed at the elucidation of nucleic acid structure-function relationships. This Feature summarizes the current state of MS-based approaches devised to overcome the limitations of traditional techniques and to advance different facets of nucleic acids research.