The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.