The prevalence of overweight and obesity has increased dramatically in recent decades in both children and adults, reaching an epidemic proportion. In order to gain new insights into the pathophysiology of obesity towards better personalized patient care, there is a critical need to develop reliable multiplex protein assays that can be easily implemented in clinical laboratories to enable precise monitoring of many hormones and inflammatory markers closely associated with obesity. Unfortunately, current clinical assays often lack of reproducibility, especially between different clinical laboratories due to the lack of assay standardization. Targeted mass spectrometry is a promising technology for providing robust multiplex assays for blood proteins. Therefore, the overall objective of this application is to develop and validate reproducible and transferable mass spectrometry-based multiplex protein assays for enabling precise quantification of a panel of obesity markers. The panel will cover multiple aspects of obesity pathophysiology, including glucose homeostasis and energy balance (e.g., insulin, leptin, and other hormones), pro-inflammatory markers (e.g., C-reactive protein), and anti-inflammatory markers (e.g., adiponectin). To facilitate full validation of the robustness and transferability of the assays, an inter-lab assay validation will be pursued. Specifically, Aim 1 will be focused on initial assay development for optimal configurations and on demonstrating confident multiplex detection of endogenous analytes in blood plasma. Aim 2 will be centered on assay optimization and full assay characterization in the aspects of reproducibility, stability, selectivity, linearity, and limit of quantification. Aim 3 will demonstrate the utility of the assays through inter-lab quantification of endogenous analytes in a pilot cohort of clinical plasma samples from normal weight and obese subjects, and obese individual before and after weight loss and benchmarking against well-established immunoassays for selected analytes such as insulin and leptin. To facilitate the easy- implementation of the validated assays in other laboratories, all assay data including chromatographic data and detailed standard operating procedures will be shared through public data repositories and assay portals. Together, the project will establish robust and easy-to-transfer multiplex assays that will enable precise monitoring of the pathophysiology of obesity.