C-peptide is a widely used measure of pancreatic beta cell function. It is produced in equimolar amounts to endogenous insulin but is excreted at a more constant rate over a longer time. Methods of estimation include urinary and unstimulated and stimulated serum sampling. Modern assays detect levels of c-peptide which can be used to guide diabetes diagnosis and management. We explore the evidence behind the various tests available. We recommend the glucagon stimulation c-peptide testing owing to its balance of sensitivity and practicality. C-peptide levels are associated with diabetes type and duration of disease. Specifically a c-peptide level of less than 0.2 nmol/l is associated with a diagnosis of type 1 diabetes mellitus (T1DM). C-peptide level may correlate with microvascular and macrovascular complications and future use of insulin therapy, as well as likely response to other individual therapies. We explore the potential uses of c-peptide measurement in clinical practice.