Drug target identification is an important premise of drug discovery and their mechanism investigation. In this study, we developed a temperature-assisted limited proteolysis-coupled mass spectrometry (TALiP-MS) method to systematically study drug binding targets and binding sites at the proteome level. We have proved that TALiP-MS was suitable for a variety of ligands, including cyclosporin A, geldanamycin and staurosporine.