Type 2 diabetes mellitus is a lifestyle‐related disease whose prevalence continues to increase worldwide. It is attributed to decreased or ineffective insulin secretion and induces hyperglycemia. The hyperglycemic state of patients with type 2 diabetes mellitus causes various quality‐of‐life‐related complications, which have become key health problems globally. However, a transformative therapy for type 2 diabetes mellitus has not yet been established, and new pharmacotherapeutics that induce insulin secretion are yet to be developed. To easily identify substances that induce endocrine secretions in the pancreas, we developed a simple, rapid, and easy experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. Levels of insulin and amylase that were released into the organ bath were measured. In our previous work, we found that insulin secretion decreased, and amylase secretion increased, over time in non‐treated groups. In the current study, insulin secretion increased in the group treated with glucagon‐like peptide‐1, a gastrointestinal hormone that induces insulin secretion. In contrast, there was no effect on amylase secretion. Trypsin activity and lactate dehydrogenase, an enzyme marker for tissue damage, were detected in the medium. To assess the effect of trypsin inhibition on insulin secretion using the organ bath technique, 1, 3, and 10 µg/mL trypsin inhibitor (TI) was added to the medium. Red mercurochrome dye was found to permeate the tissue samples, indicating that TI could also permeate the pancreas preparations. Pancreatic damage with or without TI was assessed by staining with hematoxylin and eosin and trypan blue. Hematoxylin and eosin staining revealed no significant differences in tissue structure with and without TI. However, acinar cell degeneration in the non‐treated group was slightly higher than that in the group treated with 10 µg/mL TI. Trypan blue staining revealed no apparent difference between the non‐treated group and group treated with 3 µg/mL or 10 µg/mL TI. In all TI‐treated pancreatic preparations, trypsin activity was significantly lower than that in non‐treated preparations, and the reduction in insulin secretion over time was almost completely abolished. These results suggest that the use of a pancreatic organ bath is a sensitive and reproducible method for ex vivo assessment of pancreatic function and that TI could maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.