<span class="paragraphSection"><div class="boxTitle">Abstract</div>Human transfer RNA (tRNA) anticodon variants are a source of translation error. The tRNA^Ser_AGA-2–3 variant (G35A) occurs in 2% of the human population and causes mis-incorporation of serine at phenylalanine codons. Here, we developed a dual fluorescent reporter to quantify mis-incorporation levels in live human and murine cells and validated mistranslation by mass spectrometry. In β-lymphocytes from individuals in the 1000 genomes project, we confirmed the anticipated genotype of cells with A35 minor alleles, and tRNA sequencing demonstrated expression, C32 hypo-modification, and partial 5′-fragmentation of the endogenous mutant tRNA^Ser_AAA. Nanoparticle delivery of the fluorescent reporter confirmed serine mis-incorporation in the pan-genome cell lines. The data demonstrate that a natural genome-encoded human tRNA mutant causes mistranslation in cells derived from healthy individuals. Our findings have important implications for translation fidelity in humans and the application of missense suppressor tRNAs to medicine.</span>