Glioblastoma is the most aggressive primary brain tumor with a poor mean survival even with the current standard of care. Kinase signaling analyses of clinical glioblastoma samples provide a physiologically relevant view of oncogenic signaling networks. Here, we describe the methods that enable the quantification of protein expression profiles and phosphotyrosine signaling across flash frozen and optimal cutting temperature (OCT) compound embedded tumor specimens. The data derived from these experiments can be used to identify the intra- and inter-patient heterogeneity present in these tumors. Correlation and functional analyses on the quantitative protein expression and phosphotyrosine signaling data obtained from clinical samples can be used to identify tyrosine kinase signaling networks present in these tumors and reveal the differential expression of functionally related proteins. This chapter provides the quantitative mass spectrometry methods required for the identification of in vivo oncogenic signaling networks from human tumor specimens.