To expand the software tools that enable interpretation of MS/MS spectra of modified nucleic acids in both positive and negative polarities, we have developed an R-based application developed in the Shiny framework that generates theoretical fragment ions in silico from a user-provided sequence and identifies them by their isotopic distributions directly in the m/z domain. Herein we showcase the use of this software platform, named: Shiny Application for Fragment Annotation by Relative Isotopes (Nucleo-SAFARI), on several oligonucleotides (both DNA and RNA) of varying length (10-80 nt) and highlight its utility in characterizing covalent modifications via top-down fragment ion searches.