Metabolism in the pancreatic β-cells, located in the pancreatic islets of Langerhans, controls secretion of insulin, which in turn regulates metabolism in peripheral tissues. Hence, if metabolism in these cells is perturbed, insulin secretion will be insufficient and diabetes may evolve. Investigation of metabolism is challenging, as it involves thousands of reactions, producing hundreds of different substances from even a simple sugar. During recent years, novel analytical techniques have been established that allow for measurement of hundreds of metabolites in cultured, mainly rodent, β-cells that are available at sufficiently large quantities. However, cultured cells differ from their primary counterparts and differences also exist between rodents and humans. We are now establishing new highly sensitive analytical platforms that will allow for these analyses in small quantities of isolated human islets. With these platforms we intend to generate a detailed map of human islet metabolism and its relation to insulin secretion. Moreover, we intend to examine alterations in metabolism elicited by altered circulating levels of nutrients associated with pre-diabetes, characterizing the early phase of β-cell failure.