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/ionisation 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 identified proteins is Band 3 anion transporter (SCL4A1) using LC-MS/MS and data dependant analysis on paraffin sections. SCL4A1 was validated by immunohistochemistry and was elevated in relapse tissues compared to matching ovarian cancer tissues at diagnosis in three of the four patients (p<0.05, Mann-Whitney U). SCL4A1 positivity was significantly increased in serous ovarian cancer tissues following chemotherapy treatment and relapse (P=0.0093) In addition using online databases, high SCL4A1 expression was significantly associated with reduced progression-free survival (p=0.0004) and reduced overall survival (p=0.004). In conclusion, MALDI-MSI has the potential to identify proteins associated with chemotherapy resistance, which can be used as a novel therapeutic target.