There has been a renewed appreciation for the dynamic nature of ribonucleic acid (RNA) modifications and for the impact of modified RNAs on organism health resulting in an increased emphasis on developing analytical methods capable of detecting modifications within specific RNA sequence contexts. Here we demonstrate that a DNA-based exclusion list enhances data dependent liquid chromatography tandem mass spectrometry (LC-MS/MS) detection of post-transcriptionally modified nucleosides within specific RNA sequences. This approach is possible because all post-transcriptional modifications of RNA, except pseudouridine, result in a mass increase in the canonical nucleoside undergoing chemical modification. Thus, DNA-based sequences reflect the state of the RNA prior to or in the absence of modification. The utility of this exclusion list strategy is demonstrated through the RNA modification mapping of total tRNAs from the bacteria Escherichia coli, Lactococcus lactis, and Streptomyces griseus. Creation of a DNA-based exclusion list is shown to consistently enhance the number of detected modified ribonuclease (RNase) digestion products by ∼20%. All modified RNase digestion products that were detected during standard data dependent acquisition (DDA) LC-MS/MS were also detected when the DNA-based exclusion list was used. Consequently, the increase in detected modified RNase digestion products is attributed to new experimental information only obtained when using the exclusion list. This exclusion list strategy should be broadly applicable to any class of RNA and improves the utility of mass spectrometry approaches for discovery-based analyses of RNA modifications, such as are required for studies of the epitranscriptome.