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 then evaluated through targeted quantitation by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer.