With new emerging mass spectrometry technologies, it can now be demonstrated that direct tissue analysis is feasible using matrix-assisted laser desorption/ionization (MALDI) sources. A major advantage of direct MALDI analysis is to avoid time-consuming extraction, purification or separation steps, which have the potential for producing artifacts. Direct MALDI analysis of tissue sections enables the acquisition of cellular expression profiles while maintaining the cellular and molecular integrity. With automation and the ability to reconstruct complex spectral data using imaging software, it is now possible to produce multiplex imaging maps of selected biomolecules within tissue sections. Thus, direct MALDI spectral data obtained from tissue sections can be converted into imaging maps, a method now known as MALDI-imaging. MALDI-imaging combines the power of mass spectrometry, namely exquisite sensitivity and unequivocal structural information, within an intact and unaltered morphological context. Critical improvements to increase image resolution are presented in this manuscript e.g., solvent treatment, new solid ionic matrices, gold sputtering, nickel support or laser focalization. One of the most important developments is the ability to carry out either direct MALDI analysis or MALDI imaging on paraffin tissue sections, thus opening the path to an archival “gold-mine” of existing pathology samples to proteomic analysis. These developments provide new avenues for biomarker hunting and diagnostic follow-up in the clinical setting. Further developments in MALDI-imaging of specific targets provide an added dimension, as validated disease-marker-gene RNA transcripts can be analyzed along with their translation by targeting their specific protein products or metabolites. Disease/health states will thus be closely molecularly monitored at protein and nucleic acids levels, with a single technique. Taken together, MALDI imaging will become a key tool for pathology proteomic studies.