Biofluid detectable biomarkers that originate at the site of diseased tissues would be advantageous, in that, they may provide mechanistic information concerning the manifestation and progression of the disease. Unfortunately, tissue biopsies are precious samples that can generally be acquired in small amounts due to the invasive nature of the sample collection. One of the foundations of pathological diagnosis for decades has been from formalin-fixed paraffin-embedded (FFPE) tissues, of which a vast archive exists worldwide. These tissues have also been widely used for immunohistochemistry and in situ hybridization studies examining for expression of specific proteins or transcripts. Unfortunately, the ability to analyse FFPE tissues using mass spectrometry (MS) has been essentially non-existent until recently. In this review, methods that allow the extraction of peptides from FFPE tissues and their proteomic analysis using MS are described. The ability to identify the proteins extracted from FFPE tissues allows comparative analyses that enable the potential discovery of novel biomarkers at the site of the diseased tissue.