Celastrol, a natural compound classified as a pentacyclic triterpenoid, has demonstrated efficacy in inhibiting human colorectal cancer cells' growth, adhesion, and metastasis through various signaling pathways. However, the specific protein target responsible for the effects of celastrol remains unclear, limiting its potential for further applications in colon cancer treatment and drug development. In this study, we propose a novel approach by combining a cellular thermal shift assay and pulse proteolysis techniques to identify the potential binding proteins of celastrol. Utilizing proteomic profiling on 2-dimensional electrophoresis, we successfully identified eight potential binding targets. MALDI-TOF mass spectrometry was conducted to verify these proteins as EEF2, STIP1, GAPDH, FLNA, SETSIP, GANAB, TXNDC17, and PRDX2. Our results provide valuable insights into the protein targets through which celastrol exerts its pharmacological effects, opening up new avenues for targeted therapies and drug development in colorectal cancer.