Abstract: Biomarkers are characteristics that can be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. The discovery of biomarkers is the first step in its research. Its essence is to screen out differentiated substances in different biological states or processes. In this phase, the analysis strategy focuses on high-throughput quantitative and qualitative (and/or semi quantitative) performance. Currently, the focus of biomarker research has shifted to the protein level. In the discovery of protein biomarkers, mass spectrometry has been widely used in different strategies and ways, and is still making progress. This review summarizes the common and new mass spectrometry application strategies in the discovery process of protein biomarkers.