Chemoresistance is a major therapeutic obstacle in cancer patients, and the mechanisms of drug resistance are not fully understood. In the present study, we established platinum-resistant human ovarian cancer cell lines and identified differentially expressed proteins related to platinum resistance. The total proteins of two sensitive (SKOV3 and A2780) and four resistant (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP) human ovarian cancer cell lines were isolated by two-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were identified using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). In total, 57 differential protein spots were identified, and five proteins, including annexin A3, destrin, cofilin 1, Glutathione-S-transferase omega 1 (GSTO1-1), and cytosolic NADP+-dependent isocitrate dehydrogenase (IDHc), were found to be co-instantaneous significance compared with their parental cells. The expression of the five proteins was validated by quantitative PCR and western blot, and the western blot results showed complete consistency with proteomic techniques. The five proteins are hopeful to become candidates for platinum resistance. These may be useful for further study of resistance mechanisms and screening of resistant biomarkers.