New findings in the field of epitranscriptomics have led to resurgence in the study of modified nucleic acids in RNA. Current mass spectrometry methods for the study of the epitranscriptome lack key information on the state of modified nucleotides in RNA and cellular lysates. Presented here is the first report of modified nucleotides in RNA and the cell, and provides the first reported method for the untargeted analysis of these compounds using HILIC-HRMS. We utilized a novel extraction method to measure both the bound and unbound modified nucleotides in a single sample. This method also provides separation of many modified nucleoside species. MS-FINDER software was used to provide in silico structure predictions for the creation of fragmentation rules for separation of methylated modified nucleotides on the MS2 level. Annotated here are 12 modified nucleotide species and 20 nucleosides across RNA types and cell lines.