Abstract: Target screening is a pivotal step in modern drug research and development. The hypothesis-driven biochemical assays are frequently used in traditional drug industry. While, recently, the technical advance of high-throughput and high resolution mass spectrometer, as well as the broad usage of LC-MS/MS, have facilitated the implementation of MS-based proteomic technologies in drug target screening. In spite of chemical proteomics have made dramatic breakthrough in this area, its intrinsic problems like the demand of chemical derivation on drug molecule remain an obstacle. Herein, the broadly concerned chemical modification-free approaches in the last 15 years were reviewed, including cellular thermal shift assay (CETSA), thermal proteome profiling (TPP), stability of proteins from rates of oxidation (SPROX), solvent-induced protein precipitation (SIP), drug affinity responsive target stability (DARTS), pulse proteolysis (PP), limited proteolysis (LiP). Their advantages and disadvantages were compared as well as the applications.