Background/Aim: We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. Materials and Methods: The action of TI (1-10 μg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. Results: The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. Conclusion: Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.