Ovarian cancer is the most lethal gynaecological disease, with poor 5-year survival rates and limited treatment options for patients who develop resistant disease. The majority of ovarian malignancies, up to 70% of cases, are high-grade serous carcinomas that have high chemosensitivity to first line platinum-based therapies. However, 75% of patients will become chemoresistant, following relapse. The underlying mechanism for developing resistance to chemotherapy in ovarian cancer is poorly understood. In this study, we employed peptide matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in formalin fixed paraffin embedded sections of ovarian cancer tissues at the time of diagnosis and following relapse from 4 patients with serous cancer. Using, MALDI-MSI we have identified m/z features that were present in relapsed tissues but absent in ovarian cancer tissues at diagnosis. One of these was identified as transketolase using LC-MS/MS and data dependent analysis on paraffin sections. Transketolase was validated by immunohistochemistry and was elevated by ~3 fold in relapse tissues compared to matching ovarian cancer tissues at diagnosis (P=0.035, paired t-test). In addition, transketolase (TKT) expression was significantly increased (P=0.0075, unpaired t-test) in carboplatin resistant CAOV3 cells compared to parental cells measured by qRT-PCR. In summary, MALDI-MSI has the potential to identify proteins associated with chemotherapy resistance that can be evaluated as a novel therapeutic target.