Using bottom-up oligonucleotide LC-MS/MS in combination with stable isotope labeling, chemical derivatization, and bioinformatics variable modification search, a total of 25 modifications were mapped to the sequences of 16S and 23S RNA in B. subtilis. 10 modification sites, previously identified by others using alternative methods were independently confirmed using targeted MS/MS data analysis. Modification types, namely base/ribose methylation, pseudouridines and dihydrouridine, and modification positions are fully consistent with prior experimental evidence and agree well with modifications that were annotated in E. coli and other bacteria species.