The research was conceived and fully performed at New England Biolabs. Transfer RNAs carry numerous post-transcriptional modifications that support folding, stability, and decoding accuracy. In the hyperthermophilic archaeon Thermococcus kodakarensis these modifications are expected to contribute to thermotolerance. A quantitative and comprehensive profile of tRNA modifications in T. kodakarensis has not yet been reported. Two technical factors have limited progress: (i) total cellular RNA is dominated by ribosomal RNA (rRNA, 80-90%), which can mask tRNA signals in mass spectrometry, and (ii) many methods either quantify modifications without positional information or localize modifications without robust quantification. This thesis develops a sequence-agnostic, chemoenzymatic enrichment strategy that exploits the universal 3′-CCA of mature tRNAs and is compatible with LC-MS/MS. The workflow proceeds in four steps: (1) controlled removal of the terminal adenosine (A) with Exonuclease T (ExoT), (2) re-addition of 3′-A by the Schizosaccharomyces pombe A-adding enzyme (CCA2) using an ATP analogue, (3) installation of a biotin handle via copper-free click chemistry, and (4) streptavidin capture to yield an enriched tRNA pool suitable for mass spectrometry. Method development at analytical inputs (~120 ng tRNA) revealed that CCA2 shows strong fidelity for ATP, whereas ExoT efficiently removes terminal ATP but does not cleave 2′-azido-2′-deoxyadenosine once installed. Leveraging these observations, a one-pot co-incubation reaction with ExoT and CCA2 was used to favor analogue incorporation while countering trace ATP, generating a substantial proportion of labeled tRNA for downstream tagging. Applied to Escherichia coli’s small fraction of total RNA (<200-nt) and purified MRE600 tRNA, the workflow produced enriched pools that showed the expected azido tagging and biotinylation mass shift by intact-mass LC-MS, discrete pulldown bands on microfluidic electrophoresis, and nucleoside profiles characteristic of tRNA. Extension to Thermococcus kodakarensis total RNA achieved analogous enrichment with intact-mass analysis and microfluidic electrophoresis confirmation. A comprehensive assessment of tRNA-species bias and site-resolved mapping remains future work. Overall, this thesis provides a practical, probe-free route to enrich mature tRNAs from complex mixtures at low input while maintaining compatibility with downstream LC-MS/MS. The results lay the groundwork for quantitative characterization of archaeal tRNA modification landscapes and offer a general template that can be adapted to other systems whose tRNAs bear a 3′-CCA terminus.