We describe a strategy for tracking Mg²⁺-initiated folding of ³²P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA₁(Gln) reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of ³²P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.