Glioblastoma multiforme (GBM) is the most aggressive World Health Organization grade IV brain tumor. With median survival rate of less than 15 months, GBM accounts for more than half of all neoplasm of the brain. While therapies for GBM patients consist of maximal resection of the affected area followed by extensive radiochemotherapy, clinical prognosis remains poor due to resistance to these treatments. The present study investigates the regulation of key proteins as well as proteolytic cleavage events in non-malignant tissues of the brain, early stages from astrocytomas (grade I, II and III), and GBM grade IV. Multiplexed analyses of extracted proteins from clinically dissected human tissues were carried out using isotopic labeling followed by two-dimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 5609 non-redundant proteins were identified, many of which are known to be involved in the up regulation of proteolysis and the down regulation of neuronal functions such as synaptic transmission and neurological system processes. To elucidate proteolytic processing in astrocytomas and GBM tissues, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess cleavage events in disease progression. Comparative analyses identified a total of 10,227 protein N-termini in GBM and non-malignant tissues, and approximately 5300 non-redundant protein N-termini in the comparison of low grade astrocytomas and GBM tissues. Differential cleavage processes were identified in a number of extracellular matrix proteins including actin, tubulin, glial fibrillary acidic protein and microtubule-associated proteins. Taken together, our study provides an in-depth mass spectrometry based profiling of proteolytic events in the progression of early stages of astrocytomas to GBM. Our study is a first step towards “clinical degradomics”.