Accurate assessment of the (111)In-exendin-3 uptake within the pancreas requires exact delineation of the pancreas, which is highly challenging by MRI and CT in rodents. In this study, the pancreatic tracer (99m)Tc-demobesin-4 was evaluated for accurate delineation of the pancreas to be able to accurately quantify (111)In-exendin-3 uptake within the pancreas. Healthy and alloxan-induced diabetic Brown Norway rats were injected with the pancreatic tracer (99m)Tc-demobesin-4 ([(99m)Tc-N4-Pro(1),Tyr(4),Nle(14)]bombesin) and the beta cell tracer (111)In-exendin-3 ([(111)In-DTPA-Lys(40)]exendin-3). After dual isotope acquisition of SPECT images, (99m)Tc-demobesin-4 was used to define a volume of interest for the pancreas in SPECT images subsequently the (111)In-exendin-3 uptake within this region was quantified. Furthermore, biodistribution and autoradiography were performed in order to gain insight in the distribution of both tracers in the animals. (99m)Tc-demobesin-4 showed high accumulation in the pancreas. The uptake was highly homogeneous throughout the pancreas, independent of diabetic status, as demonstrated by autoradiography, whereas (111)In-exendin-3 only accumulates in the islets of Langerhans. Quantification of both ex vivo and in vivo SPECT images resulted in an excellent linear correlation between the pancreatic uptake, determined with ex vivo counting and (111)In-exendin-3 uptake, determined from the quantitative analysis of the SPECT images (Pearson r = 0.97, Pearson r = 0.92). (99m)Tc-demobesin-4 shows high accumulation in the pancreas of rats. It is a suitable tracer for accurate delineation of the pancreas and can be conveniently used for simultaneous acquisition with (111)In labeled exendin-3. This method provides a straightforward, reliable, and objective method for preclinical beta cell mass (BCM) quantification with (111)In-exendin-3.