Chemical biology tools play a crucial role in target discovery by enabling the identification and validation of biological molecules involved in disease pathways. Small molecule probes are used to modulate protein function and study cellular responses. Activity-based protein profiling (ABPP) allows selective labelling of active enzymes within complex proteomes. Click chemistry facilitates biorthogonal labelling and visualization of biomolecular interactions. CRISPR-Cas9 and RNAi technologies are combined with chemical tools for functional genomics studies. Photo affinity labelling helps identify drug-binding targets through covalent capture upon light activation. These tools bridge chemistry and biology, accelerating the understanding of disease mechanisms. Ultimately, they guide the development of more effective and selective therapeutics.