Transfer RNA (tRNA) modifications have emerged as critical posttranscriptional regulators of gene expression that impact diverse biological and disease processes. While there is extensive knowledge about the enzymes installing the dozens of tRNA modifications – the tRNA epitranscriptome – very little is known about how metabolic, signaling and other networks integrate to regulate tRNA modification levels. Here we took a comprehensive first step at understanding epitranscriptome regulatory networks by developing a high-throughput tRNA modification profiling platform and applying it to a Pseudomonas aeruginosa transposon insertion mutant library comprising over 5,000 strains. Analysis of the more than 200,000 tRNA modification data points validated annotation of hundreds of tRNA modification related genes, uncovered novel tRNA modifying enzymes, and revealed tRNA modification regulatory networks in P. aeruginosa.