The ability to detect 2′-O-methylation sites (Nm) in high-throughput fashion is important, as increasing evidence points to a more diverse landscape for this RNA modification as well as the possibility of yet unidentified functions. Here we describe an optimized version of RibOxi-seq, which is built upon the original published method, that not only accurately profiles ribosomal RNA (rRNA) Nm sites with minimal RNA input but is also robust enough to identify mRNA intronic and exonic sites.