When researchers design specific ligands to regulate target proteins in cellular regulation or drug discovery, they usually focus on a higher affinity of ligands. Consequently, ligands having lower affinities are frequently removed in the ligand selections. However, these selections do not necessarily explain the mechanism of inhibitory actions of ligands. Therefore, we have doubt that the high affinity selection is always equal to the specificity selection. Since the specific ligands possess a specific binding manner in the protein-ligand interactions, we focus on the biophysical quality, especially thermodynamics, of ligand interactions but not high affinity to obtain the specific ligands against the target proteins. Thermodynamic analysis using Isothermal Titration Calorimetry (ITC) is one of the representative biophysical methods, since it can monitor the direct binding of compounds with thermal reaction and discuss the binding specificity. We are studying some ligand screenings for target enzymes, for example, we obtained one hit small molecule to inhibit the target using biophysical methods including thermodynamic analysis. This ligand successfully inhibits the target protein in cells, although its binding affinity is around μM range. Therefore, we propose that the important factor governing the ligands’ specificities is not only high affinity but also the thermodynamics.