Background Abnormal secretion of insulin and glucagon is a fundamental element in the pathogenesis of diabetes. Subjects with type 1 diabetes have severely impaired β-cell function at time of diagnosis, and several strategies to preserve β-cell function have been tested in clinical trials using plasma C-peptide responses as the primary endpoint. In type 2 diabetes, hyperglycemia results from inappropriate glucagon secretion and an imbalance between an insufficient and delayed insulin response during meals and insulin resistance. Thus, analysis of β-cell function and insulin resistance are of major importance for understanding the underlying pathophysiology of glucose metabolism in people with diabetes and the mechanisms of action of pharmacological treatments. Analysis of α-cell function has received less attention, but availability of glucagon-like peptide (GLP)-1 receptor agonists and glucagon receptor antagonists have clearly demonstrated the importance of glucagon for fasting hyperglycemia. Although glucagon plasma profiles are clearly abnormal in type 2 diabetes, α-cell function per se is not necessarily perturbed. The aim of the present chapter is to discuss optimal tests for understanding the impact of pharmacological and other treatments on α-and β-cell function in subjects with type 1 or type 2 diabetes. Key Methods Currently-applied tests of tests of α-and β-cell function based on secretion of their products after an overnight fast are evaluated. Dynamic tests assessing the responses of α-and β-cells to meals and oral or intravenous glucose +/-non-glucose secretagogues will be discussed. The range of different techniques for the assessment of insulin secretion and response to therapeutic interventions reflects the absence of a gold standard reference method. No single method adequately captures all potentially relevant aspects of β-and α-cell function. Selection of the most appropriate tests should thus be guided by the purpose of the study, e.g. evaluation of direct effects of secretagogues, or complex mechanisms involving for instance activation of the incretin axis. Several test methods may be required to provide a comprehensive understanding that is sufficient for making drug development decisions. Assessing the integrity of the incretin axis and α-cell function has come to prominence with the introduction of dipeptidyl peptidase (DPP-4) inhibitors and GLP-1 receptor agonists as well as development of glucagon receptor antagonists.