In the last two decades, an incredible amount of effort has been put forth to gain a molecular level understanding of human pathophysiology through genome sequencing projects and human proteome projects. Despite that, there is a lot more to discover with respect to complex molecular and biochemical networks in cellular and tissue-specific functions which are largely mediated by proteins and their modifications. With the advent of the post-genomic era, scientists have developed an inherent interest in interdisciplinary approaches and accredited proteomics studies with powerful chemical probe molecules as a result of carefully designed synthetic chemistry. Chemoproteomics is one such tool that provides an opportunity to screen interactions between small molecule drugs and proteins in their native cellular environments and simultaneously understand the preceding mechanism of action. This chapter discusses how chemoproteomics has helped us evolve from phenotype-based drug discovery to target-based drug discovery by shedding light on the different methods for the identification and validation of protein targets interacting with the small molecule drugs. Given its contribution to the field of drug discovery and biomarker identification, it is apt to discuss the importance of chemoproteomics in the field of biotechnology and the advancements it has brought to the pharmaceutical sciences © 2023 Munishwar Nath Gupta.