Exploring protein targets of drugs and deciphering the specific mechanisms-of-action at the molecular level of these interactions are crucial steps in the development of drugs to explore target related phenotypes and to treat human diseases. We have developed target protein identification methods including conventional affinity chromatography using labeled small molecules as well as recent methods using label-free small molecules such as Drug Affinity Responsive Target Stability (DARTS) and Cellular Thermal Shift Assay (CESTA) in combination with LC-MS/MS analysis to identify the direct binding proteins of drugs with phenotypic activities in angiogenesis and autophagy. The direct interaction between drug and the target protein is validated via bio-physical, and bio-informatics methods. Moreover, biological relevancy of this “drug-target” interaction is verified through genetic modulation that facilitates structure based better drug development. In this presentation, our studies on target identification of drug-target interaction for exploring new mechanism studies toward phenotypes of interest and translational applications will be presented by introducing our case studies of protein target identification and validation of natural products and clinical drugs perturbing autophagy.