Personalized medicine relies upon individualized diagnosis that provides molecular information delineating optimal therapeutic strategies. For many diseases, but especially cancer, the development of predictive biomarkers requires performing assays directly on the diseased tissue or tumor. The last decade has seen the explosion of both prognostic and predictive biomarkers in the research setting, but few of these biomarkers have entered widespread clinical use. This article examines issues concerning tissue-biomarker development and the hurdles faced in reaching the goal of truly personalized medicine. Targeted therapy guided by predictive biomarkers is possible; however, for significant progress, researchers need to focus on three key issues: robust assays for the clinic, validation in clinically relevant environments and assuring appropriate analytes are available for these new assays.