In the present study, we applied UVPD and CAD to narrow 2 m/z windows across an MS1 spectrum of a desalted yeast tRNA extract in a gas-phase fractionation approach. The specific isolation windows were selected to cover the region of the MS1 spectrum with the most abundant precursor ions. Charge-reduced EPD product ions observed in the UVPD spectra were identified by their observed isotopic distributions compared to theoretical isotope patterns modelled based on a database of characterized yeast tRNA sequences. Once precursor ions were identified in a UVPD spectrum, their theoretical fragment ions were identified in a parallel CAD spectrum acquired for the same 2 m/z region in the MS1 spectrum. Additionally, to assess the confidence with which the identified tRNAs were characterized by MS/MS, we implemented a scoring calculation -analogous to the P-score commonly employed in top-down proteomics experiments -to provide a consistent metric by which top-down MS characterization can be assessed across each spectrum and tRNA isoform.