Non-coding RNAs (ncRNAs) have emerged as important regulatory elements of gene expression in a wide variety of biological processes. RNA molecules mature through various post-transcriptional processing events in a spatiotemporal manner. Post-transcriptional modifications (or RNA editing) are characteristic structural features of RNA molecules and are required for their proper functioning. Most of these modifications have been found in abundant RNA molecules, such as tRNAs, rRNAs and/or UsnRNAs. It has recently been reported that even small RNAs are modified. In mammals, certain populations of miRNAs (~6%) contain inosine (I)1,2. In plants, the 3'-termini of miRNAs are modified by 2'methylation3, and this modification is required for normal maturation of miRNAs4. Recently, we found that mouse Piwi-interacting RNAs (piRNAs) also have 3'-terminal 2'methylations5. Direct analysis of RNA molecules by mass spectrometry provides qualitative information about the nature of the modifications embedded in RNA molecules, such as base modifications and terminal chemical structures. Here, we describe a detailed protocol for the mass spectrometric analysis of the 3' termini in ncRNAs.