This study aims to optimize a reversed-phase high-performance liquid chromatography (RP-HPLC) method for developing an oral semaglutide formulation and to evaluate interactions between semaglutide and various excipients. Two columns with different pore sizes were used to assess detection sensitivity and repeatability. The separation efficiency was compared using standard solutions, and optimal analytical conditions were established. Comparison of the two columns showed that the large column exhibited superior sensitivity and repeatability. Thus, this column was used for further optimization. Analytical Condition A was set with a detection wavelength of 250 nm and a mobile phase of 0.01 N KH₂PO₄:Acetonitrile (ACN) = 70:30 (v/v). Analytical Condition B was set with a detection wavelength of 250 nm, applying a gradient elution with mobile phase A (deionized water (DIW) + 0.05% trifluoroacetic acid (TFA)) and mobile phase B (ACN + 0.05% TFA). Condition B exhibited superior separation of Semaglutide and excipients compared to Condition A. The final optimized method employed an Agilent C18 (5 μm, 4.6 × 150 mm) column as the stationary phase, with a column oven temperature of 30 °C, a flow rate of 0.5 mL/min, an injection volume of 10 μL, and an analysis time of 30 minutes. These findings are expected to contribute to providing a reliable analytical method from the pre-formulation to the formulation stage in the development of oral semaglutide formulations