Post-transcriptional RNA modification is now recognized as a major player of gene expression regulation, even if the exact roles of such modifications are not yet clearly established at the molecular level. Further advancements in this field are however still hindered by la imited knowledge on the presence and exact positions of these modifications in RNAs. Several high-throughput methods for precise mapping of m5C, m6A and pseudouridine residues have been reported during the last 4 years, but many RNA modifications still escape detection and identification. Recently, a method for high-throughput mapping of 2'-O-Me has been proposed, but its application is still limited due to a rather unconventional library preparation protocol. In this study we adapted the published protocol in order to use standard and commercially available library preparation protocol as well rather popular Illumina sequencing for 2'-O-Me mapping. The approach was successfully applied to model yeast rRNA, and further extended for analysis of human rRNA modification. Screening of various human cell lines indicate that a subset of 2'-O-Me are constitutively modified independently from conditions and used cell lines, while other show considerable differences, allowing regulation of translation at the ribosome's properties level. Further studies are required to link this differential rRNA modification to human diseases, as well to understand regulation of mRNA translation by differentially methylated ribosomes.