Riboswitches are intricate, metabolite-binding RNA structures found in the non-coding regions of mRNA. Allosteric changes in the riboswitch that are induced by metabolite binding are harnessed to control the genes of a variety of essential metabolic pathways in eubacteria and in some eukaryotes. In this chapter, we describe an RNA structure analysis technique called “in-line probing.” This assay makes use of the natural instability of RNA to elucidate secondary structure characteristics and ligand-binding capabilities of riboswitches in an entirely protein-free manner. Although this method has most frequently been used to examine riboswitches, the structures of any RNAs can be examined using this technique.