Pseudouridine (Ψ) is the most abundant posttranscriptional RNA modification; yet little is known about its prevalence and function in messenger RNA, mostly due to the challenges in the transcriptome-wide detection of Ψ. Here, we report CeU-Seq-a selective chemical labeling and pull-down method for the comprehensive analysis of transcriptome-wide pseudouridylation; this sequencing method will hopefully pave the way for functional studies of Ψ-mediated biological regulation in the future.