Exploring the interaction of a small molecule with its target protein is pivotal to understand the cellular mechanism by which the small molecule acts. However, discovering the on- or off-target proteins of small molecule is often the most challenging and time-consuming step. Chemical proteomics has played as a key research engine to identify direct interacting proteins and to explore mechanisms of action of small molecules towards functional and translational applications. A number of methodologies including conventional affinity chromatography using labeled small molecules as well as recent target identification methods with label-free small molecules such as Drug Affinity Responsive Target Stability (DARTS), Stability of Proteins from Rates of Oxidation (SPROX), Cellular Thermal Shift Assay (CETSA), and Thermal Proteome Profiling (TPP) have been developed and applied to identify the direct binding proteins of small molecules. This interaction information of small molecule and target protein facilitates structure based better drug development and functional annotation of target protein as well. Furthermore, integration of MALDI-MS imaging technology with chemical proteomics will enable to validate the interaction of label-free small molecule with target protein in tissue that harnesses the preclinical studies of small molecules in respect with their efficacy, toxicity, and pharmacokinetics. In this presentation, recent advances of chemical proteomics for target identification of small molecules towards functional and translational applications will be presented by introducing our case studies.