The in-depth analysis of the ADME profiles of drug candidates using in vitro models is essential for drug development since a drug’s exposure in humans depends on its ADME properties. In contrast to efforts in developing human in vitro absorption models, only a limited number of studies have explored models using rats, the most frequently used species in in vivo DMPK studies. In this study, we developed a monolayer model with an effective barrier function for ADME assays using rat duodenal organoids as a cell source. At first, we developed rat duodenal organoids according to a previous report, but they were not able to generate a confluent monolayer. Therefore, we modified organoid culture protocols and developed cyst-enriched organoids; these strongly promoted the formation of a confluent monolayer. Furthermore, adding valproic acid to the culture accelerated the differentiation of the monolayer, which possessed an effective barrier function and apicobasal cell polarity. Drug transporter P-gp function as well as CYP3A activity and nuclear receptor function were confirmed in the model. We expect our novel monolayer model to be a useful tool for elucidating drug absorption processes in detail, enabling the development of highly absorbable drugs.