N¹-methyl adenosine (m¹A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m¹A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m¹A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m¹A can notably modulate RNA structure. It also blocks Watson–Crick–Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m¹A. In this study, we introduce a reduction-based m¹A sequencing (red-m¹A-seq). We report that NaBH₄reduction of m¹A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m¹A to m⁶A to provide good controls, allowing the detection of m¹A with higher sensitivity and accuracy. We applied red-m¹A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m¹A sites, but also new m¹A sites in mt-tRNA^Asn-GTTand 5.8S rRNA.