It has long been known that pseudouridine (Ψ) is the most abundant modified nucleotide in stable RNAs, including tRNA, rRNA, and snRNA. Recent studies using massive parallel sequencing have uncovered the presence of hundreds of Ψs in mRNAs as well. In eukaryotes and archaea, RNA pseudouridylation is introduced predominantly by box H/ACA RNPs, RNA-protein complexes each consisting of a single RNA moiety and four core proteins. It has been well established that Ψ plays an essential role in regulating the structure and function of stable RNAs in several model organisms, including yeast, Xenopus laevis, and humans. However, the functional role of Ψ in mRNA remains to be elucidated. One possibility (and true for stop/termination codons) is that Ψ influences decoding during translation. It is imperative, therefore, to establish a system, in which one can site-specifically introduce pseudouridylation into target mRNA and biochemically test the impact of mRNA pseudouridylation on protein translation. Here, we present a method for (1) site-specific conversion of uridine into Ψ in mRNA by designer box H/ACA RNP, (2) detection of Ψ in target mRNA using site-specific labeling followed by nuclease digestion and thin layer chromatography, and (3) analysis of recoding of pseudouridylated premature termination codon in mRNA during translation.