Update information. Hepatocellular carcinoma (HCC) is susceptible to early recurrence and lacks effective predictive markers. Mass Spectrometry (MS)-based proteomics has emerged as a potent tool for identifying cancer biomarker profiles in various bodily fluids. In this study, we established a MS-based biomarker discovery and validation workflow. Firstly, the workflow successfully identified a total of 447 serum proteins in 179 individuals (126 HCC patients and 53 liver cirrhosis (LC)) using the Data Independent Acquisition-MS (DIA-MS) strategy. Then, candidate serum biomarkers were validated in 192 individuals (142 HCC patients and 50 LC patients) using Parallel Reaction Monitoring (PRM) strategy.