Mast cells (MCs) are innate immune cells that can cause allergic reactions. Pseudo-allergic reactions are a type of reaction that have the clinical symptoms of an allergy without involving the IgE pathway of immune MCs. Some studies have found that the MRGPRX2 is crucial for mediating such allergic reactions, as some antagonists and agonists have the same binding sites. One promising direction is to use the endogenous agonist of the receptor as a competitive inhibitor through structural modifications. Substance P induces mast cell activation through the MRGPRX2. Therefore, we designed and synthesized a new substance P analogue, and a β-hexaminosidase assay and Ca²⁺ mobilization assay were performed to evaluate the anti-allergic effect of the substance P analogue, while its binding characteristic to MRGPRX2 was analyzed by molecular docking and cell membrane chromatography. The results suggest that the substance P analogue could be a potential therapeutic agent for managing pseudo-allergic reactions.