MALDI-mass spectrometry imaging (MALDI-MSI) is a technique that allows proteomic information, that is, the spatial distribution and identification of proteins, to be obtained directly from tissue sections. The use of in situ enzymatic digestion as a sample pretreatment prior to MALDI-MSI analysis has been found to be useful for retrieving protein identification directly from formalin-fixed, paraffin-embedded (ffpe) tissue sections. Here, an improved method for the study of the distribution and the identification of peptides obtained after in situ digestion of fppe pancreatic tumor tissue sections by using MALDI-mass spectrometry imaging coupled with ion mobility separation (IMS) is described. MALDI-IMS-MS images of peptide obtained from pancreatic tumor tissue sections allowed the localization of tumor regions within the tissue section, while minimizing the peak interferences which were observed with conventional MALDI-TOF MSI. The use of ion mobility separation coupled with MALDI-MSI improved the selectivity and specificity of the method and, hence, enabled both the localization and in situ identification of glucose regulated protein 78 kDa (Grp78), a tumor biomarker, within pancreatic tumor tissue sections. These findings were validated using immunohistochemical staining.