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Genome-wide dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) is a method suitable for the in vivo investigation of RNA folding on a genome-wide scale. Based on the RNA structure-specific in vivo chemical modification by DMS and the encoding of resultant modifications as mismatches during reverse transcription, DMS-MaPseq produces ratiometric RNA structure data that does not require base-by-base correction to control samples. Due to its high signal-to-noise ratio, DMS-MaPseq is the preeminent choice amongst mutational profiling techniques. Here we describe the genome-wide DMS-MaPseq protocol, which produces libraries suitable for Illumina sequencing and can be applied across many model systems.