An integrated two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) system was applied to investigate the proteome from the whole serum sample without abundant protein depletion. An integrated HPLC column containing both a strong-cation-exchange section and a C18 reverse-phase section was used for online 2D LC separation, which was coupled to a linear ion trap mass spectrometer. Over a thousand protein groups were identified from a single normal serum sample. This method enabled two-dimensional separation of tryptic peptides from complex biological samples using a conventional one-dimensional chromatography system. The serum sample from a patient with hepatocellular carcinoma (HCC) was also analyzed, and the gold standard HCC biomarker alpha-fetoprotein (AFP) was detected. Collectively, the integrated 2D LC-MS/MS system was proved to be an efficient tool to analyze the serum proteome.