Chemoresistance is a poor prognostic factor in breast cancer and, thus, presents a significant clinical challenge. The mechanisms of chemoresistance involve multiple complex biological processes. This study aims to identify common contributory factors to chemoresistance in breast cancer by comparing protein expression profiles of chemosensitive MCF-7 breast cancer cells and cells resistant to two different commonly used anti-cancer drugs (adriamycin and paclitaxel). Expression of the ATP binding cassette transporter, P-glycoprotein (P-gp), in breast tumours has previously been found to correlate with poor prognosis in vivo and, accordingly, we confirmed overexpression of P-gp in both adriamycin- and paclitaxel-resistant MCF-7 cells. Using two-dimensional gel electrophoresis and MALDI-TOF peptide mass fingerprinting, we identified 20 proteins differentially expressed between chemosensitive, adriamycin-resistant and paclitaxel-resistant MCF-7 cells. Cytokeratin-8, keratin-19, Hsp-27, 14-3-3 epsilon, annexin-A2 and phosphoglycerate kinase-1 showed altered expression in both adriamycin- and paclitaxel-resistant cells. Validation of a number of these changes was confirmed by Western blotting. Our findings provide further insights into the complex mechanisms of chemoresistance, as well as representing an attractive starting point for the identification of potential protein biomarkers to predict response to chemotherapy in breast cancer in vivo.