Organoids from intestinal regions have proven to be useful tools to study intestinal epithelial responses to different conditions. Roux-en-Y gastric bypass (RYGB) has been associated with important intestinal adaptations, but the mechanisms underlying these changes are still poorly understood. Organoids could therefore be used to better decipher the intestinal adaptations associated with this surgery. Rat is a common model to assess RYGB responses in vivo , but surprisingly, very few studies managed to develop organoids from rat small intestine. The primary objective of this study was to establish a protocol for cultivating organoids derived from the small intestine of healthy rats. The second objective of this study focuses on the development of organoids from the small intestine of rats subjected to RYGB to evaluate whether phenotypic or gene expression differences emerge. We successfully devised a functional protocol for developing organoids from fresh or frozen rat small intestine tissues. The obtained organoids exhibit significant variability, making interpretation challenging. Variability is observed in size, shape, and the number of organoids developed from the same sample, but also gene expression, depending on samples prepared on different days or from fresh or frozen tissues. This protocol was then applied to the small intestine of RYGB or sham-operated rats. However, we did not detect any major difference in size between intestinal organoids derived from Sham rats and those from RYGB rats. The expression of several genes (peptide transporters, amino acid transporters, genes specific to certain types of intestinal cells, etc.) was also assessed, and inter-experiment variability was higher than any effect due to the operation on the rat the intestinal tissue was originating. In conclusion, this study has established a functional protocol to grow small intestine organoids in rats. Initial results suggest that in our experimental conditions, organoids obtained from rats subjected to RYGB do not differ from those obtained from Sham rats. However, increasing the sample size and improving reproducibility between experiments will be essential to confirm these findings.