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The identification of novel small molecules that promote pancreatic beta-cell proliferation is an important approach to therapeutic discovery for diabetes. Because human islets are not easy to culture, and attach poorly to plates, it had not been feasible to run high-throughput phenotypic assays in these cells. Therefore, most laboratories have turned to rodent islets for ease of culture and accessibility. However, rodent islets are not physiologically similar to human islets, either in terms of islet architecture or endocrine cell interactions within the islet, and data generated in rodent islets do not typically translate to human islet biology. To address this challenge, we developed a human islet culture system for high-throughput screening using a thymidine analog, EdU, to detect beta-cell replication during screening. Simultaneous monitoring of EdU incorporation and beta cell numbers provides a robust assay for beta-cell replication, and is now becoming a standard protocol enabling screening in human islets.