Reversible and dynamic RNA modifications play important roles in fine-tuning gene expression. N6, 2′-O-dimethyladenosine (m⁶Am), a terminal modification at mRNA cap, mediates various biological effects. However, limitations of the current m⁶Am detection methods lead to a lack of potential applications. Here, we describe a specific and sensitive method, termed m⁶Am-seq, that can detect m⁶Am at single-base resolution. m⁶Am-seq is based on optimized in-vitro demethylation assay and RNA immunoprecipitation, which can distinguish m⁶Am from 5′-UTR m⁶A. We provide a step by step protocol to perform m⁶Am-seq, including experimental procedures and sequencing data analysis. Collectively, we describe m⁶Am-seq, a robust tool to reveal both m⁶Am and 5′-UTR m⁶A methylome, enabling further functional and mechanistic study of m⁶Am modification.