The most frequently employed activation method employed for MS/MS analysis of RNA is collisionally activated dissociation (CAD), which has been shown to routinely yield abundant terminal w-, c-, and y-type fragments and high sequence coverage for individual homogeneous RNAs. For heterogeneous populations of RNA where precursor ions overlapping in the m/z domain may be co-isolated and co-activated, CAD may result in chimeric spectra, confounding identification. Although modern fragment identification workflows can identify well-resolved fragments in heterogeneous spectra, confident characterization of each RNA, including sequences and modifications, can be difficult without exact knowledge of the identities of the precursor species that were initially isolated prior to MS/MS. Ultraviolet photodissociation (UVPD) can overcome this obstacle by providing additional information about the composition of the precursor ion population along with production of additional diagnostic fragment ions, complementing characterization by CAD. Upon UV photoactivation, negatively charged nucleic acids generally yield a series of high-abundance electron photodetachment (EPD) products. These EPD products are intact precursor ions that have lost one or more electrons and exhibit lower charge states than the original precursor, resulting in an array of charge-reduced precursors distributed across a higher m/z region. In an MS/MS experiment undertaken on a population of overlapping precursors of different charge states, the dispersed charge-reduced EPD product ions can be more easily identified by their nowresolved isotopic distributions which were previously overlapping. This process is analogous to charge-reduction workflows in proteomics, which enable analysis of heterogeneous precursor and fragment populations, often via ion-ion reactions in the gas phase. Extending this chargereduction methodology to tRNA analysis facilitates identification of intact, charge-reduced precursors in UVPD spectra and subsequent assignment of corresponding fragment ions in the CAD spectra of the same m/z isolation window.