In neurodegenerative diseases, including pathologies with well-known causative alleles, genetic factors that modify severity or age of onset are not entirely understood. We recently documented the unexpected prevalence of transfer RNA (tRNA) mutants in the human population, including variants that cause amino acid mis-incorporation. We hypothesized that a mistranslating tRNA will exacerbate toxicity and modify the molecular pathology of disease-causing alleles and investigated the combinatorial effects of tRNA variants in cellular models of Huntington’s disease. This dataset represents MS/MS data confirming Phenylaline to Serine amino acid misincorporation events caused by the tRNA-Ser G35A variant described in our study. The wildtype (WT) or mistranslating tRNA (VAR) were expressed in murine N2a cells along with mCherry protein. We affinity purified the mCherry protein to search for Ser misincorporation events at Phe codons.