Advances in mass spectrometry, proteomics, protein bioanalytical approaches, and biochemistry have led to a rapid evolution and expansion in the area of mass spectrometry-based biomarker discovery and development. The last decade has also seen significant progress in establishing accepted definitions, guidelines, and criteria for the analytical validation, acceptance, and qualification of biomarkers. These advances have coincided with a decreased return on investment for pharmaceutical research and development and an increasing need for better early decision making tools. Empowering development teams with tools to measure a therapeutic interventions impact on disease state and progression, measure target engagement, and to confirm predicted pharmacodynamic effects is critical to efficient data-driven decision making. Appropriate implementation of a biomarker or a combination of biomarkers can enhance understanding of a drugs mechanism, facilitate effective translation from the preclinical to clinical space, enable early proof of concept and dose selection, and increase the efficiency of drug development. Here we will provide descriptions of the different classes of biomarkers that have utility in the drug development process as well as review specific, protein-centric, mass spectrometry-based approaches for the discovery of biomarkers and development of targeted assays to measure these markers in a selective and analytically precise manner.