Mistranslation, the mis-incorporation of an amino acid not specified by the “standard” genetic code, occurs in all cells. tRNA variants that increase mistranslation arise spontaneously and engineered tRNAs can achieve mistranslation frequencies approaching 10% in yeast and bacteria. The goal of this study was to detect and quantify mistranslation from a serine tRNA variant with proline UGG anticodon and G26A secondary mutation engineered in yeast