Riboswitches are RNA sensors that change conformation upon binding small molecule metabolites, in turn modulating gene expression. Our understanding of riboswitch regulatory function would be accelerated by a high-throughput, quantitative screening tool capable of measuring riboswitch-ligand binding. We introduce a microfluidic mobility shift assay that enables precise and rapid quantitation of ligand binding and subsequent riboswitch conformational change. In 0.3% of the time required for benchtop assays (3.2 versus 1020 min), we screen and validate five candidate SAM-I riboswitches isolated from thermophilic and cryophilic bacteria. The format offers enhanced resolution of conformational change compared to slab gel formats, quantitation, and repeatability for statistical assessment of small mobility shifts, low reagent consumption, and riboswitch characterization without modification of the aptamer structure. Appreciable analytical sensitivity coupled with high-resolution separation performance allows quantitation of equilibrium dissociation constants (K-d) for both rapidly and slowly interconverting riboswitch-ligand pairs as validated through experiments and modeling. Conformational change, triplicate mobility shift measurements, and K-d are reported for both a known and a candidate SAM-I riboswitch with comparison to in-line probing assay results. The microfluidic mobility shift assay establishes a scalable format for the study of riboswitch-ligand binding that will advance the discovery and selection of novel riboswitches and the development of antibiotics to target bacterial riboswitches.