DNA molecules utilize adenine, thymine, cytosine, and guanine for coding genetic information. In addition to these four canonical nucleobases, DNA molecules also contain a variety of modified nucleobases that can control and regulate gene expression and chromosome structure. Elucidating the functions of DNA modifications relies on the sensitive detection, accurate quantification, and genome-wide mapping of these modifications in genomic DNA. The significant advances of techniques and methods in recent years have enabled the discovery and functional studies of a number of new modifications in DNA in both prokaryotes and eukaryotes. Mass spectrometry-based methods for analyzing DNA modifications have substantially advanced over the past decade, which has greatly stimulated the research of DNA epigenetic modifications. The emergence of next-generation sequencing technology provides complementary methodology to enable genome-wide mapping of modifications, which is very important to reveal the biological roles of DNA modifications. This perspective highlights the recent methodologies for the assessment of DNA modifications with focus on mass spectrometry and sequencing analytical strategies.