Nucleophilic amino acids play important roles in maintenance of protein structure and function, covalent modification of such amino acid residues by therapeutic agents is an efficient way to treat human diseases. Most of current clinical drugs are structurally limited to α,β-unsaturated amide as an electrophilic warhead. To alleviate this issue, many novel electrophiles have been developed in recent years that can covalently bind to different amino acid residues and provides a unique way to interrogate proteins, including “undruggable” targets. With an activity-based protein profiling (ABPP) approach, the activity and functionality of a protein and its binding sites can be assessed. This facilitates an understanding of protein function, and contributes to the discovery of new druggable targets and lead compounds. Meanwhile, many novel inhibitors bearing new reactive warhead were developed and displayed remarkable pharmaceutical properties. In this perspective, we have reviewed the recent remarkable progress of novel electrophiles and their applications in target identification and drug discovery.