Inosine (I), a modified base found in the double-stranded regions of RNA in metazoans, has various roles in biological processes by modulating gene expression. Inosine is generated from adenosine (A) catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes in a process called A-to-I RNA editing. As inosine is converted to guanosine (G) by reverse transcription, the editing sites can be identified by simply comparing cDNA sequences with the corresponding genomic sequence. One approach to screening I sites is by deep sequencing based on A-to-G conversion from genomic sequence to cDNA; however, this approach produces a high rate of false positives because it cannot efficiently eliminate G signals arising from inevitable mapping errors. To address this issue, we developed a biochemical method to identify inosines called inosine chemical erasing (ICE), which is based on cyanoethylation combined with reverse transcription. ICE was subsequently combined with deep sequencing (ICE-seq) for the reliable identification of transcriptome-wide A-to-I editing sites. Here we describe a protocol for the practical application of ICE-seq, which can be completed within 22 d, and which allows the accurate identification of transcriptome-wide A-to-I RNA editing sites.