Genetic information encoded in genomic DNA is transcribed to mRNAs and then the codons on mRNA are decoded by transfer RNAs (tRNAs) during protein synthesis. tRNAs deliver amino acids to ribosomes and proteins are synthesized from the amino acids on the ribosomes according to the decoded genetic information. Therefore, tRNA plays a key role during the translation of genetic information. tRNAs contain numerous modified nucleosides, which regulate the accuracy and efficiency of protein synthesis. Modified nucleosides in tRNA are synthesized by tRNA modification enzymes. Therefore, unveiling the mechanisms by which tRNA modification enzymes selectively recognize substrate tRNAs from non-substrate RNAs; the when, where, and how many tRNAs are being modified by the modification enzymes, is of crucial importance to understand the protein synthesis machinery. Addressing these key questions is, however, challenging due to the lack of a high-throughput technique that identifies the characteristic properties of tRNA modification enzymes.