Since drug resistance is a complex and multifactorial event involving activation/repression of multiple biochemical pathways, we used a proteomic approach to study cisplatin resistance and drug response in human tumor cell lines. The cervix squamous cell carcinoma cell line A431 and its cisplatin‐resistant subline, A431/Pt, were used as a model system. The experimental set‐up involved not just a two‐way comparison of the control vs. the drug‐resistant cell line, but also an acute cisplatin treatment of both cell lines, leading to a four‐way comparison, as follows: 1) A431 vs. A431/Pt cells; 2) A431 vs. A431 cisplatin exposed cells; 3) A431/Pt vs. A431/Pt cisplatin exposed cells; 4) A431 cisplatin exposed cells vs. A431/Pt cisplatin exposed cells. We found modulation of proteins, which could be classified under various categories, such as molecular chaperones (e.g. heat‐shock proteins HSP60, HSP90, HSC71, heat‐shock cognate 71 kDa protein), Ca2plus;‐binding proteins (e.g. calmodulin, calumenin), proteins involved in drug detoxification (such as peroxiredoxins PRX 2 and PRX 6, and glutathione‐S‐transferase, GST), anti‐apoptotic proteins (such as 14‐3‐3 switched on in cisplatin‐exposed cells) and ion channels (such as VDAC‐1, voltage‐dependent anion‐selective channel). In particular, the basal levels of HSC71 and HSP60 were increased in A431/Pt cells as compared to A431 cells, and cisplatin exposure resulted in up‐regulation of HSP60 and HSP90 only in A431 cells. Moreover, cisplatin exposure up‐regulated the anti‐apoptotic 14‐3‐3 protein in both cell lines, GST in sensitive cells and PRX6 in A431/Pt cells. These findings are consistent with a constitutive expression of defence factors by resistant cells and with activation by cisplatin of mechanisms acting to protect cells from drug‐induced damage. This pattern of response, also observed in parental cells, could reflect an intrinsic resistance of this tumor type.