Riboswitches are regions of mRNA that bind selectively certain metabolites, thereby regulating gene expression. We have developed a method, which uses a combination of biophysical techniques (equilibrium dialysis, waterLOGSY and T2 relaxation-edited NMR spectroscopy and isothermal titration calorimetry) that allows screening and identification of novel ligands for riboswitches. By this method a library of 1300 fragments was screened on the E. colithiamine pyrophosphate (TPP) riboswitchthiM, resulting in the identification of 17 hits. The compounds showed KD values from 22 to 670 μM, with four compounds having KD values <100 μM. The fragments are structurally diverse and their ligand efficiency indicates good prospects for structure-guided elaboration. In addition, a riboswitch functional assay based on in vitrotranscription translation (IVTT) of the reporter gene luciferase was developed. This assay system is an alternative to in vivo assays and constitutes a valuable tool to determine the effect of small molecules on riboswitch-regulated gene expression.