The introduction of technologies such as mass spectrometry and protein and DNA arrays, combined with our understanding of the human genome, has enabled simultaneous examination of thousands of proteins and genes in single experiments, which has led to renewed interest in discovering novel biomarkers for cancer. The modern technologies are capable of performing parallel analyses as opposed to the serial analyses conducted with older methods, and they therefore provide opportunities to identify distinguishing patterns (signatures or portraits) for cancer diagnosis and classification as well as to predict response to therapies. Furthermore, these technologies provide the means by which new, single tumor markers could be discovered through use of reasonable hypotheses and novel analytical strategies. Despite the current optimism, a number of important limitations to the discovery of novel single tumor markers have been identified, including study design bias, and artefacts related to the collection and storage of samples. Despite the fact that new technologies and strategies often fail to identify well-established cancer biomarkers and show a bias toward the identification of high-abundance molecules, these technological advances have the capacity to revolutionize biomarker discovery. It is now necessary to focus on careful validation studies in order to identify the strategies and biomarkers that work and bring them to the clinic as early as possible.