Knowledge about β-cell mass and/or function could be of importance for the early diagnosis and treatment of diabetes. However, measurement of β-cell function as an estimate of β-cell mass is currently the only method possible in humans. The present study was performed to investigate different functional tests as predictors of β-cell mass in the Göttingen minipig. β-cell mass was reduced in the Göttingen minipig with a combination of nicotinamide (100 [n = 6], 67 [n = 25], 20 [n = 2], or 0 mg/kg [n = 4]) and streptozotocin (125 mg/kg). Six normal pigs were included. An oral glucose tolerance test (OGTT) (n = 43) and insulin secretion test (n = 30) were performed and pancreata obtained for stereological determination of β-cell mass. During OGTT, fasting glucose (r² = 0.1744, P < 0.01), area under the curve for glucose (r² = 0.2706, P < 0.001), maximum insulin secretion (r² = 0.2160, P < 0.01), and maximum C-peptide secretion (r² = 0.1992, P < 0.01) correlated with β-cell mass. During the insulin secretion test, acute insulin response to 0.3 g/kg (r² = 0.6155, P < 0.0001) and 0.6 g/kg glucose (r² = 0.7321, P < 0.0001) and arginine (67 mg/kg) (r² = 0.7732, P < 0.0001) and maximum insulin secretion (r² = 0.8192, P < 0.0001) correlated with β-cell mass. This study supports the use of functional tests to evaluate β-cell mass in vivo and has established a validated basis for developing a mathematical method for estimation of β-cell mass in vivo in the Göttingen minipig.