Chemoresistance remains the major barrier to effective treatment of ovarian cancer and predicting chemoresponse is an unmet clinical need. We developed a carboplatin resistant ovarian cancer cell line model and observed enhanced migratory and invasive capabilities in resistant cells. Proteomics analysis was able to separate these populations based on their molecular features. However, the comparable analysis of patients’ samples did not show a clear distinction between samples from sensitive and resistant tumours, highlighting the interpatient variability. To be able to monitor direct response of patient derived samples to chemotherapy, we used patient derived ovarian cancer spheroids (3-dimensional multicellular cell clusters), which can be easily isolated from malignant ascites. We performed MALDI-mass spectrometry imaging on spheroids and were not only able to visualise three distinct layers (proliferating, quiescent, and necrotic) within the spheroids, but were also able to monitor drug response. For potential translation into a clinical setting, we developed an assay ready tissue culture plate, which needs minimal sample handling of malignant ascites and can be preloaded with chemotherapy combinations of choice. The chemotherapy response of the patient derived cells/spheroids can be easily monitored using standard fluorochromic stains.