Disease specific proteins are highly valuable as clinical biomarkers, which can be used in early diagnosis, monitoring disease progressions and evaluating therapies. The identification and characterization of protein biomarkers in different physiological and pathological sources of interest under diverse milieu represent a key area of clinical proteomics. However, owing to the inherent complexities and the large dynamic ranges of proteins, there are many practical issues and challenges in discovering the low-abundance, disease-specific biomarkers from heterogeneous tissues and biofluids. Thus, an integrated approach for selective protein pre-fractionation, purification and separation, is necessary to detect low-abundant biomarkers in proteomics research. This review will summarize recent advancements in clinical sample preparation for removing high abundant proteins, as well as the improved two-dimensional gel electrophoresis- and mass spectrometry-based technologies for resolving low-abundant proteins. We will also elaborate the proteomic strategies for targeting protein sub-populations with special interests, such as high molecular weight protein complexes, membrane or its associated proteins, and organelle specific proteins etc. We will emphasize the use of these strategies to interrogate the current proteomic researches in clinical biomarker discovery.