Many issues have been found to be addressed for the interactions between drugs and their target proteins in the small molecule drug discovery, e.g. low efficiency of lead construction and off-target effect with low specificity; these are due to limited physicochemical information on the drug-protein interactions. In the true molecular target-based drug discovery, hit, seed, and lead compounds should show high binding affinity and specific inhibition activity. To obtain the correct interaction profiles of small compounds with a target protein, technical development of physicochemical approaches (biophysical methods) for the interactions is essential for the next generation molecular target-based drug discovery. 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. Calorimetric analysis using ITC has recently been an attractive approach in the fields of the small molecule drug discovery. In this review, our recent studies for the small-molecule drug screening were introduced, where the inhibitors for target proteins were validated using ITC. Furthermore, we demonstrate that ITC analysis is the potential selection of validated hits and lead compounds in PPI screening.