A major scientific challenge at the present time for cancer research is the determination of the underlying biological basis for cancer development. It is further complicated by the heterogeneity of cancer's origin. Understanding the molecular basis of cancer requires studying the dynamic and spatial interactions among proteins in cells, signaling events among cancer cells, and interactions between the cancer cells and the tumor microenvironment. Recently, it has been proposed that large-scale protein expression analysis of cancer cell proteomes promises to be valuable for investigating mechanisms of cancer transformation. Advances in mass spectrometry technologies and bioinformatics tools provide a tremendous opportunity to qualitatively and quantitatively interrogate dynamic protein-protein interactions and differential regulation of cellular signaling pathways associated with tumor development. In this review, progress in shotgun proteomics technologies for examining the molecular basis of cancer development will be presented and discussed.