CpG methylation of DNA is an epigenetic marker that is highly related to the regulation of transcription initiation. For analysis of CpG methylation in genomic DNA sequences, bisulfite-induced modification in combination with polymerase chain reaction (PCR) is usually utilized, but it cannot be straightforwardly applied to methylated short- and middle-sized DNAs, such as < 500 base pairs (bp), which are often utilized in structural biology studies. In the present study, we applied nano-electrospray ionization mass spectrometry (nano-ESI-MS) for the characterization of methylated DNA with < 400 bp prepared in vitro. First, double-stranded DNA oligomers were methylated with recombinant M.SssI DNA methylase, which has been reported to modify completely and exclusively CpG sites in the sequence. The fragments generated by the digestion with methylation-insensitive restriction nuclease were then analyzed to identify the methylation levels by nano-ESI-MS, without liquid chromatography (LC) separation. By methylation-insensitive nuclease digestion, we divided the DNA strands into several fragments, and nano-ESI-MS enabled the accurate analysis of methylation levels in the DNA fragments with a relatively small amount of DNA sample prepared under optimized conditions. Furthermore, it was revealed that M.SssI methylase hardly modifies the CpG sites closely positioned at the ends of linear DNA. The present method is similar to the strategy for post-translational modification analysis of proteins and is promising for the rapid and definitive characterization of methylated DNA that may be used in structural biology studies. Electronic supplementary materialThe online version of this article (10.1007/s13361-019-02304-5) contains supplementary material, which is available to authorized users.