Transfer RNAs play a critical role in protein synthesis by matching mRNA codons to their corresponding amino acids. Their post-transcriptional modifications, which modulate structure, stability, and codon decoding properties, are now recognized as key regulators of translation and cellular adaptation across diverse organisms, including bacterial pathogens. Here, we provide a comprehensive analysis of tRNA modifications in Staphylococcus aureus using extensive oligonucleotide mass spectrometry, generating a high-confidence modification map for each individual tRNA species, including the non-proteogenic tRNAGly.