Oligonucleotide (ON) or RNA mapping using liquid chromatography coupled to mass spectrometry (LC-MS) has recently emerged as an attractive approach to support the development of nucleic acid-based medicines, in particular mRNA. This technique provides valuable information on the primary structure of mRNA by combining enzymatic digestion, high-resolution chromatographic separation, accurate mass determination, optimized fragmentation strategies and advanced data treatment, similar to the peptide mapping workflows commonly employed for structural elucidation and quality assessment of therapeutic proteins. This review critically examines the latest developments in enzymatic digestion methods, liquid chromatography techniques, mass spectrometry conditions, and data treatment strategies to improve the performance of ON mapping and also provides some perspectives to further optimize this workflow.