The intestinal permeability of mitragynine was investigated in situ using a single pass intestinal perfusion (SPIP) absorption model, in small intestine of rat using mitragynine in the absence/presence of the permeability markers, P-gp and/or CYP3A4 inhibitors. Mitragynine demonstrated high intestinal permeability (Peff of 1.11 × 10-4 cm/s) that is in the range of highly permeable drugs such as propranolol (Peff of 1.27 × 10-4 cm/s) indicating that it readily crosses the intestine. The addition of azithromycin (P-glycoprotein inhibitor) and ciprofloxacin (CYP3A4 inhibitor) or combination of both has no effect on intestinal permeability of mitragynine across the rat small intestine.