Associating changes in protein levels with the onset of cancer has proven as an effective approach to identify clinically relevant diagnostic biomarkers. In the present study, we used sera from 205 patients representing two cohorts for biomarker discovery using label-free proteomic analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We performed first untargeted proteomic analysis of sera to identify candidate proteins with statistically significant difference between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. These proteins were later on evaluated through targeted quantitation by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. The results of this study illustrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to investigate changes in protein levels and discover novel diagnostic biomarkers.