<p>Transfer RNAs (tRNAs) are essential for decoding mRNAs into proteins and are increasingly recognized as dynamic regulators of gene expression. Their function is shaped by intricate layers of post-transcriptional processing, modification, aminoacylation, and fragmentation, all of which have been implicated in human disease. Recent advances in high-throughput sequencing have transformed our ability to profile tRNAs and their associated modifications, uncovering their roles in cancer, neuronal function, immune response, and stress response. In this review, we summarize emerging tRNA sequencing technologies and highlight how these approaches reveal fundamental insights into tRNA regulation and its therapeutic potential.</p>