Mass spectrometry (MS)-based profiling of clinical specimens has been increasingly used in cancer research to characterize changes in protein expression between tumor and healthy tissue or between the blood of diseased and healthy individuals. These molecular profiles may lead to distinct insights that are not readily evident using in vitro cultured cells or animal models and may facilitate discovery of clinically applicable biomarkers and novel drug targets. However, the discovery of valid cancer biomarkers and drug targets using MS-based proteomics has proven difficult, primarily due to the analytical challenges exemplified in the wide dynamic range of human plasma protein levels (i.e., > 10 orders of magnitude) and the fact that the 10 most abundant proteins constitute ~ 90% of the total plasma protein mass. Analytical challenges related to MS-based profiling of clinical samples obtained from diverse patient populations are complex, different from those in basic cancer research characterized by controlled experimental conditions employing in vitro cultured cells or transgenic animals. Therefore, experimental design and sample preparation represent the most challenging steps within a clinical proteomic workflow aimed at the development of reproducible and high-throughput methods for cancer biomarker and drug targets discovery. This presentation focuses on our recent advances in experimental design and method development using high resolution/accuracy MS-based proteomics for molecular profiling of clinical specimens in the context of cancer biomarker and novel drug targets research/discovery.