Degradation-Based Protein Profiling (DBPP) is a novel strategy combining PROTAC technology with quantitative proteomics and immunoprecipitation-mass spectrometry (IP-MS) to identify multiple targets of natural products. This study applied DBPP to celastrol, a triterpenoid compound from traditional Chinese medicine, to identify its targets. Using a PROTAC toolbox, the researchers identified known targets such as IKKβ, PI3Kα, and CIP2A, as well as potential new targets like CHK1, OGA, and ERCC6L. The first glycosidase degrader was also developed. DBPP relies on non-covalent protein-protein interactions rather than protein-small molecule interactions, enabling the identification of proteins with moderate or weak binding affinities. The strategy was validated using western blot, co-immunoprecipitation, and cellular thermal shift assays, confirming that celastrol interacts directly with IKKβ, PI3Kα, and OGA. CHK1 was identified as a potential toxicity target, while ERCC6L was found to be another possible target. The study also demonstrated the effectiveness of mixed-toolbox quantitative proteomics in improving the efficiency and cost-effectiveness of target identification. Overall, DBPP provides a powerful tool for identifying targets of natural products and other drug molecules, potentially accelerating drug discovery and development. The integration of quantitative proteomics with IP-MS allows for the identification of target proteins that form ternary complexes and undergo degradation, enhancing the accuracy of target identification. The study highlights the potential of DBPP in complementing existing chemical proteomics technologies and advancing the pharmaceutical field.Degradation-Based Protein Profiling (DBPP) is a novel strategy combining PROTAC technology with quantitative proteomics and immunoprecipitation-mass spectrometry (IP-MS) to identify multiple targets of natural products. This study applied DBPP to celastrol, a triterpenoid compound from traditional Chinese medicine, to identify its targets. Using a PROTAC toolbox, the researchers identified known targets such as IKKβ, PI3Kα, and CIP2A, as well as potential new targets like CHK1, OGA, and ERCC6L. The first glycosidase degrader was also developed. DBPP relies on non-covalent protein-protein interactions rather than protein-small molecule interactions, enabling the identification of proteins with moderate or weak binding affinities. The strategy was validated using western blot, co-immunoprecipitation, and cellular thermal shift assays, confirming that celastrol interacts directly with IKKβ, PI3Kα, and OGA. CHK1 was identified as a potential toxicity target, while ERCC6L was found to be another possible target. The study also demonstrated the effectiveness of mixed-toolbox quantitative proteomics in improving the efficiency and cost-effectiveness of target identification. Overall, DBPP provides a powerful tool for identifying targets of natural products and other drug molecules, potentially accelerating drug discovery and development. The integration of quantitative proteomics with IP-MS allows for the identification of target proteins that form ternary complexes and undergo degradation, enhancing the accuracy of target identification. The study highlights the potential of DBPP in complementing existing chemical proteomics technologies and advancing the pharmaceutical field.