One of the most popular methods for figuring out a drug’s intestinal permeability is the intestinal perfusion (SPIP) approach. Metoprolol as tartrate and phenol red are frequently utilized as reference compounds in perfusion tests to assess the permeability coefficient of the study’s focus component, Domperidone maleate. Our study’s goal was to create and validate a reversed-phase liquid chromatographic technique for the simultaneous detection of Domperidone maleate, metoprolol, and phenol red for use in intestinal perfusion studies. The analysis was performed on a C18 column 150 × 1.5 mm, 5.0 µm using a mobile phase consisting of methanol: 20 mM potassium dihydrogen phosphate buffer 43:57, v/v; pH 3.1. The procedure was validated using the FDA’s standards for stability, linearity, precision, and accuracy. All calibration curves were linear r2> 0.99. The system suitability parameters of the method are the required limits all analytes. The in situ intestinal permeability experiments can easily be standardized with the help of this newly created and proven approach.