Immune checkpoint immunotherapy (ICI) is a promising treatment of cancer patients suffering from e.g. non-small cell lung cancer (NSCLC). However, this therapy is only successful in 25-30% of treated patients and associated with severe immune-related events. Therefore, (protein) biomarkers predicting ICI success are needed. Proteomic approaches, in particular mass spectrometry, have already proven their relevance in discovery of new biomarkers, allowing early diagnosis or prediction of therapy response resulting in a better patient’s quality of life and improvement of health care. We focus on the combinatorial use of mass spectrometry imaging (MSI) with top-down proteomics on NSCLC tissues in an attempt to better understand the communication between immune cells and NSCLC cells and to provide new crucial (pathogenic) insights. By uncovering the role of these protein markers in anti-tumor responses, we strengthen the value of our biomarkers and aim towards a new lead for anticancer (immune)therapy.