Hallmarks of malignancy can be monitored by protein signatures in serum or plasma. The current challenge in cancer research is the identification of clinically reliable protein biomarkers for diagnostic and prognostic purposes. A widely used and powerful technique to screen tumor markers is two-dimensional gel electrophoresis (2DE). This review provides an overview of 2DE functionality with its advantages and drawbacks as well as a current literature overview of gel-based cancer biomarker discovery in serum/plasma. In this context, 11 of the 12 studies reviewed here identified at least one of eight classical serum or high-abundant proteins (HAPs). Expression levels of those proteins are regulated by a vast variety of different physiological, metabolic and immunological stimuli leading to a questionable application as cancer-specific markers. Misinterpretation of HAPs as tumor markers might be caused by either the experimental setup or the technical and analytical potential in gel-based serum or plasma proteomics to detect low-abundant proteins, or a combination thereof. Additionally, based on currently available technology we propose an optimized experimental workflow to allow detecting cancer-specific protein markers of low abundance in future 2DE studies.