Updated publication reference for PubMed record(s): 30990045. Updated publication reference for PubMed record(s): 30990045. Although thousands of intact proteins can be feasibly identified in recent years, global quantification of intact proteins is still challenging. Herein, we develop a high-throughput strategy for global intact protein quantification based on chemical isotope labeling. The isotope incorporation efficiency is as high as 99.2% for complex intact protein samples extracted from HeLa cells. Further, the pTop 2.0 software is developed for automated quantification of intact proteoforms in a high-throughput manner. The high quantification accuracy and reproducibility of this strategy have been demonstrated for both standard and complex cellular protein samples. A total of 2,283 intact proteoforms originated from 660 protein accessions are successfully quantified under anaerobic and aerobic conditions and the differentially expressed proteins are observed to be involved into the important biological processes such as stress response.