Abstract The objective of this 4-year R01 project is to develop an innovative and comprehensive toolkit, including a collection of fluorescent protein (FP) based biosensors, a microfluidic Islet-On-a-Chip, a smartphone fluorescence microscope, and an imaging analysis application, optimized for the potency evaluation of human islets as cell-based therapy in Type 1 diabetes (T1D), as well as for characterization of the phenotype and physiology of emerging new biologics for future T1D treatment. We propose to use genetically encoded fluorescent biosensors to evaluate key physiological parameters of β- cells and islets, and furthermore, to define the potency, phenotype, and physiology of various promising biologics for T1D cell replacement therapy. Leveraging the complementary expertise of our strong team and our extensive preliminary results, we will pursue the following two Specific Aims: 1. Develop and optimize fluorescent biosensors to monitor key physiological processes in β-cells and islets. 2. Integrate biosensors with microfluidic arrays, a smartphone fluorescence microscope, a customized imaging analysis application for standardized, high-content characterization of islet biologics. The outcome of this project will be an economic and integrated system for convenient and reliable characterization of human islets, xenogeneic islets, encapsulated islets, and stem-cell-derived β-cells. These in vitro assays, which innovatively evaluate several key physiological parameters of β-cells and islets, can not only be used for standardization and quality control, but also be important screening tools to improve the generation, storage, and transport of next-generation biologics. Our proposed work will generate significant impacts on development of current and future cell replacement therapy for diabetes.