Recent years have seen a burst in the number of studies investigating tRNA biology. With the transition from a gene-centred to a genome-centred perspective, tRNAs and other RNA polymerase III transcripts surfaced as active regulators of normal cell physiology and disease. Novel strategies removing some of the hurdles that prevent quantitative tRNA profiling revealed that the differential exploitation of the tRNA pool critically affects the ability of the cell to balance protein homeostasis during normal and stress conditions. Furthermore, growing evidence indicates that the adaptation of tRNA synthesis to cellular dynamics can influence translation and mRNA stability to drive carcinogenesis and other pathological disorders. This review explores the contribution given by genomics, transcriptomics and epitranscriptomics to the discovery of emerging tRNA functions, and gives insights into some of the technical challenges that still limit our understanding of the RNA polymerase III transcriptional machinery.