Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has improved over the years and is increasingly being used for biomarker discovery directly from human tissue sections. State-of-the-art technology currently enables a resolution down to 20 μm. MSI therefore allows the correlation of spatial and temporal protein expression profiles with distinct morphological features without requiring target-specific reagents, such as antibodies. Several studies have demonstrated the strength of the technology for uncovering new markers that correlate with disease severity as well as prognosis and therapeutic response. This review provides an overview of MALDI imaging functionality and its advantages and disadvantages, and provides a current literature overview of malignancy-based biomarker detection. Further improvements on instrumentation sensitivity, image processing and sample preparation will enable the detection of novel, tissue-specific biomarkers. However, emphasis should be given to large validation studies and/or subsequent identification of differentially observed protein peaks in order to transfer MSI protein profiling and/or novel biomarkers thereof into clinical use.