A detailed understanding of how protein kinase signalling is perturbed in human cancers requires network-level interrogation. Our laboratory has established cutting-edge technology platforms in mass spectrometry-based proteomics that provide global insights into how tyrosine phosphorylation and the expressed kinome are altered in specific cancer subtypes. Application of these approaches has led to insights such as characterization of the signalling network characteristics of triple negative breast cancer cells, identification of mechanisms of docetaxel resistance in castrate-resistant prostate cancer, definition of novel subtypes of pancreatic cancer, and mapping of global kinome responses to specific oncogenes. Overall this strategy is identifying novel therapeutic targets and biomarkers for particular poor prognosis human cancers.