Classic histopathological examination of tissues remains the mainstay for cancer diagnosis and staging. However, in some cases histopathologic analysis yields ambiguous results, leading to inconclusive disease classification. We set out to explore the diagnostic potential of mass spectrometry-based imaging for tumour classification based on proteomic fingerprints. Combining mass spectrometry with supervised machine learning, we were able to distinguish colorectal tumor from normal tissue with an overall accuracy of 98%. In addition, this approach was able to predict the presence of lymph node metastasis in primary tumour of endometrial cancer with an overall accuracy of 80%. These results highlight the potential of this technology to determine the optimal treatment for cancer patients to reduce morbidity and improve patients’ outcomes.