Over the last decade we have witnessed tremendous advances in our understanding of the underlying molecular alterations in human cancer. This has stimulated excitement for our ability to develop effective targeted therapies and diagnostic tests based on genetic information. However, there have been only limited successes, to a great extent due to the challenges of matching inferred signaling pathway alterations based on specific genetic mutations with often inferred effects of a particular drug. The presentation will provide an example of a functional, pathway-based approach for identification of drug-tailored, efficacy-predictive biomarkers based on post-translationally modified proteins involved in the aggressive, metastatic cancer phenotype. Moreover, we will describe the de novo development and clinical validation of a novel proteomics prostate cancer biopsy test for intact tissue based on quantitative multiplex immunofluorescence and image analysis. The described approaches enable development of powerful prognostic and efficacy-predictive, protein-based, quantitative biomarkers for better clinical treatment decisions and outcome.