The identification of angiogenesis-related proteins is important for the development of new anti-angiogenetic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins which are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomic techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by matrix-assisted laser desorption\ionization Fourier transform mass spectrometry (MALDI-FTMS). Subsequently, the samples were fractionated by nano-LC prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). This combined approach enabled us to identify four proteins which appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms and reactive conditions in which neo-angiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.