Objective: Microvascular invasion (mVI), a major predictive factor of tumoral recurrence and mortality in patients with hepatocellular carcinoma (HCC), is only detectable on pathological examination. So far, there is no reliable tool to identify mVI prior to surgical procedures. MALDI Imaging Mass Spectrometry (IMS) represents a new analytical tool to provide the relative abundance and distribution of the whole proteins expressed in a tissue section. The aim of this study was to compare, using MALDI IMS, the tissue proteome of HCC without and with mVI in order to identify surrogate biomarkers of mVI. Method: A total of 56 HCC samples obtained from surgical specimens, for which frozen samples were available, were retrospectively collected. Two groups of tumors were defined (26 HCC without mVI; 30 HCC with mVI) and were analysed by MALDI IMS. A statistical comparative analysis of acquired mass spectra was then performed in order to identify protein peaks differentially expressed between the two groups. Results: 30 protein peaks were differentially expressed between the two groups, all overexpressed in HCC with mVI. Protein characterization of some of these peaks is in progress. Conclusion: These results highlight the potential of MALDI IMS to uncover new biomarkers in liver carcinogenesis. The identification of mVI biomarkers would be helpful in the therapeutic strategy of patients with HCC.