Drug resistance is a major impediment to the successful treatment of human cancers, including ovarian cancer. Vinblastine (VLB), an antimicrotubule agent, is one of the chemotherapeutic drugs that exhibit resistance in ovarian cancer patients. To determine the protein factors that are involved in vinblastine resistance in human ovarian cancer cells, a combination of sample pre-fractionation and high-resolution 2-DE proteomic analysis was performed. Approximately 1200 proteins were detected and quantitatively compared in both nuclear/membrane and cytosolic fractions. Sixty-nine proteins from the nuclear/membrane fraction showed altered expression levels, whereas 59 were altered in the cytosolic fraction between SKOV3 (vinblastine-sensitive) and SKVLB (vinblastine-resistant) cell lines. These proteins include membrane-associated, chromatin remodeling, cytoskeletal, and microtubule-associated proteins as well as others that regulate signal transduction. This study not only demonstrates a novel understanding of the mechanism of drug resistance but also provides a valuable resource for future studies on drug resistance to vinblastine. In addition, it also represents a good example of how to increase the protein dynamic range and reduce sample complexity using currently available tools.