Queuosine (Q) in humans is a microbiome-dependent modification in the wobble anticodon position of tRNA, tRNA, tRNA, and tRNA. These tRNAs share a 343536 anticodon consensus. In humans, the Q base in tRNA and tRNA is further glycosylated to generate galactosyl-Q (galQ) and mannosyl-Q (manQ) modifications. Q-tRNA modification is known to regulate translation in a codon dependent manner, but the function of Q glycosylation is unknown. A sensitive and quantitative detection method for Q-glycosylation in tRNA is essential to investigate its biological function. Although LC/MS was used in the characterization of glyco-Q tRNA, the requirements of large amount of input material and LC/MS expertise limit its application. We recently developed an acid denaturing gel and Northern blot method to sensitively detect galQ and manQ-tRNA modification and quantify their modification fractions using just microgram amounts of total RNA. This method uses the same acid denaturing gel system for separating charged from uncharged tRNA; however, deacylated, galQ and manQ modified tRNAs are also separated from unmodified tRNAs because of the positive charge carried by the secondary amine and the large chemical moiety of the glyco-Q base. Our method enables rapid investigation of glycosylated Q modification in tRNA, and also has the potential to investigate other large tRNA modifications that carry a positive charge under acid denaturing gel conditions.