<span class="paragraphSection"><div class="boxTitle">Abstract</div>Transfer RNAs (tRNAs) are utilized by the ribosome to decode the nucleic acid alphabet. tRNA structure, stability, aminoacylation efficiency, and decoding efficacy are governed by their extensive post-transcriptional modifications. In most studies, individual tRNAs are generated using <span style="font-style: italic;">in vitro</span> transcription, which produces tRNAs devoid of these critical site-specific modifications, negatively affecting translation yields and fidelity. To address this challenge, we have developed a purification method that couples tRNA overexpression to DNA hybridization-based purification. Using this approach, we produced native tRNAs from <span style="font-style: italic;">Escherichia coli</span> in high yield and purity while retaining their complement of native post-transcriptional modifications and translational activity. We extend this technique to the purification of <span style="font-style: italic;">Mj-</span>$tRNA_{CUA}^{Opt}$ and <span style="font-style: italic;">Ma-</span>$tRNA_{CUA}^{Pyl}$, tRNAs of critical importance for genetic code expansion. We confirmed that both <span style="font-style: italic;">Mj-</span>$tRNA_{CUA}^{Opt}$ and <span style="font-style: italic;">Ma-</span>$tRNA_{CUA}^{Pyl}$ contain native <span style="font-style: italic;">E. coli</span> post-transcriptional modifications and provide the first complete modification profiles of each. Moreover, we found that <span style="font-style: italic;">in vivo-</span>generated <span style="font-style: italic;">Mj-</span>$tRNA_{CUA}^{Opt}$ and <span style="font-style: italic;">Ma-</span>$tRNA_{CUA}^{Pyl}\ $significantly outperform their <span style="font-style: italic;">in vitro-</span>generated counterparts in a