AbstractIn order to understand the molecular mechanisms of multidrug resistance (MDR) in ovarian cancer, we employed the proteomic approach of isobaric tags for relative and absolute quantification (iTRAQ), followed by LC‐MS/MS, using the cisplatin‐resistant COC1/DDP cell line and its parental COC1 cell line as a model. A total number of 28 proteins differentially expressed were identified, and then the differential expression levels of partially identified proteins were confirmed by Western blot analysis and/or real‐time RT‐PCR. Furthermore, the association of PKM2 and HSPD1, two differentially expressed proteins, with MDR were analyzed, and the results showed that they could contribute considerably to the cisplatin resistance in ovarian cancer cell. The differential expression proteins could be classified into eight categories based on their functions, that is, calcium binding proteins, chaperones, extracellular matrix, proteins involved in drug detoxification or repair of DNA damage, metabolic enzymes, transcription factor, proteins related to cellular structure and proteins relative to signal transduction. These data will be valuable for further study of the mechanisms of MDR in the ovarian cancer. J. Cell. Biochem. 109: 625–633, 2010. © 2010 Wiley‐Liss, Inc.