Abstract: OBJECTIVE Establish a method for simultaneous determination of adulteration cinnabar dye scarlet 808 and Rhodamine B By LC-MS/MS. METHODS Using Agilent ZORBAX SB-C₁₈(2.1 mm×50 mm, 1.8 μm) column, mobile phase of acetonitrile-0.1% formic acid, the flow rate was 0.3 mL·min^-1, column temperature was 30 ℃, ethanol as an extraction solvent, Ionization mode was ESI, detection mode was MRM. Selected ion pair: ①Scarlet 808 m/z 368.1→275.1, m/z 368.1→219.1, m/z 368.1→77.1; ②Rhodamine B m/z 443.2→399.2, m/z 443.2→355.0. RESULTS the linear range of A and B was 10~200 ng·mL^-1 and 9.69~193.8 ng·mL^-1 in this method; detection limit was 2 ng·mL^-1 and 1.9 ng·mL^-1; recoveries were 97.86% and 97.34%, RSD was 0.70% and 0.73%; precision, stability and repeatability of RSD were less than 4%. CONCLUSION The method of extraction is simple, determination is accurate, confirmatory high, high sensitivity, determination red dye Scarlet 808 and Rhodamine B are applicable to cinnabar, and also provide a reference basis for the safety testing of other Chinese medicine with red color.