RNA macromolecules are of increasing interest as potential drug targets. This new approach offers opportunities for urgently needed novel antibiotics, medicines against rare trinucleotide repeat disorders like Huntington’s disease and spinocerebellar ataxia or also in tumor therapy. However, conventional computational tools for structure-based drug design like molecular docking are based and optimized to predict protein-ligand interactions. The transfer from protein-ligand to RNA-ligand predictions needs proper validation as RNA and proteins show huge differences in their overall shape, charge, aromaticity and flexibility. In this project we aim to validate protein-based virtual screening tools for RNA-ligand docking and highlight pitfalls and considerations to make when searching for ligands of potential RNA drug targets. We further use a riboswitch aptamer-domain as a model system, not only to demonstrate the general suitability of molecular docking for RNA targets, but further elucidate binding thermodynamics and kinetics of RNA-ligand interactions in vitro with isothermal titration calorimetry (ITC), microscale thermophoresis (MST) and the novel switchSENSE® technology. Applying our computational and biophysical methods, general guidelines on how to design RNA-binding small molecules will be established.