RNAs are drawing increasing attention as potential therapeutic targets. A significant challenge in the RNA drug discovery process is identification of compounds that not only disrupt the natural functions of RNA by binding with high affinity, but also do so selectively. Assessing the binding mode of small molecules with RNA is important for understanding how they select their binding site and impart their mechanism of action. A number of complementary assays are often employed for analysis of the binding mode and to determine selectivity. One important technique that gives information about the binding affinity and stoichiometry is electrospray ionization mass spectrometry (ESI MS). More recent methods have also revealed the usefulness of ESI MS in determining the binding loci of small molecules on RNA. © 2019 Elsevier Inc. All rights reserved.