Electrophoretic mobility shift assay is a simple, rapid, and sensitive technique to analyze the RNA–RNA interaction. A 32 P-labeled RNA is incubated with another unlabeled RNA and subjected to electrophoresis on a native polyacrylamide gel. If two RNA molecules base pair stably, the movement of the probe RNA through the gel is retarded resulting in a characteristic band corresponding to the RNA duplex. Here, we describe the methods to study the interaction of an Hfq-binding small RNA (sRNA) and its target mRNA. Although we focus on the interaction of SgrS and its target ptsG mRNA, the methods can be applied to the analysis of base pairing between any sRNAs and their targets.