Biomedicine is developing rapidly in the 21st century. Among them, the qualitative and quantitative analysis of peptide biomarkers is of considerable importance for the diagnosis and therapy of diseases and the quality evaluation of drugs and food. The identification and quantitative analysis of peptides have been going on for decades. Traditionally, immunoassays or biological assays are generally used to quantify peptides in biological matrices. However, the selectivity and sensitivity of these methods cannot meet the requirements of the application. The separation and analysis technique of liquid chromatography-mass spectrometry (LC-MS) supplies a reliable alternative. In contrast to immunoassays, LC-MS methods are capable of providing the analytical prowess necessary to satisfy the demands of peptide biomarker research in the life sciences arena. This review article provides a historical account of the in-roads made by LC-MS technology for the detection of peptide biomarkers in the past 10 years, with the focus on the qualification/quantification developments and their applications.