Transfer ribonucleic acids (tRNA) are a biologically significant class of non-coding ribonucleic acids (ncRNAs) that pose unique analytical challenges for complete characterization. Here we present a robust and simple method for the consistent and accurate identification of individual tRNAs from a pool of total tRNA obtained from cell lysate. Through this method individual isoacceptor tRNAs are identified by the detection of unique oligonucleotide sequences which arise from a single enzymatic digestion. These unique sequences can be detected by monitoring specific transitions from precursor to product ions. Thus, for any pool of known tRNA sequences including posttranscriptional modifications, targeted tandem mass spectrometry can be used for monitoring these specific transitions. The proposed method was developed and validated using a set of known tRNAs from Escherichia coli. This approach was found to identify 41 ± 2 of the predicted 47 isoaccepting tRNAs in E. coli from targeted tandem mass spectrometry using only 24 precursor m/z values. This method should be easily adapted to other bacterial systems for both genomic and posttranscriptional analysis of tRNAs, and is likely suitable for future clinical applications.