An LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations is described. A 5 min separation was developed using a 2.1 × 30 mm (i.d. x length) C18 column. The method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide matched well with reported values of these hormones.