RNA 2'-O-methylation is one of the ubiquitous nucleotide modifications found in many RNA types from bacteria, archaea, and eukarya. We and others have recently published accurate and sensitive detection of these modifications on native RNA at a single base resolution by high-throughput sequencing technologies. Relative quantification of these modifications is still under progress and would probably reduce the number of false positives due to 3D RNA structure. Therefore, here, we describe a reliable and optimized protocol for quantification of 2'-O-Methylations based on alkaline fragmentation of RNA coupled to a commonly used ligation approach followed by Illumina sequencing. For this purpose, we describe how to prepare in vitro transcribed yeast 18S and 25S rRNA used as a reference for unmodified rRNAs and to compare them to purified 18S and 25S rRNA from yeast total RNA preparation. These reconstructed rRNA mixes were combined at different ratios and processed for RiboMethseq protocol.This technique will be applicable for routine parallel treatment of biological and clinical samples to decipher the functions of 2'-O-methylations in normal and pathologic processes or during development.