Abstract: Dingkundan is a traditional Chinese medicine formula consisted of 30 herbs, such as Radix ginseng Rubra, Cervus nippon Temminck, Saffron Stigma Croci and Pseudo-ginseng. The quality control of Cervi cornu Pantotrichum is of important for maintaining the effectiveness of Dingkundan. Herein, a method of ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for specificity identification of the Cervi Cornu Pantotrichum and Rangifer tarandus Linnaeus in Dingkundan. Dingkundan samples were digested by trypsin, and the tryptic peptides were subsequently separated by using Acquity UPLC BEH C18 column (2.1 mm×100 mm×1.7 μm) with the mobile phases comprising of acetonitrile-containing 0.1% formic acid (A) and water containing 0.1% formic acid (B). The gradient started with 3%A, and increased to 8%A at 10 min, to 20%A at 20 min, to 50%A at 30 min with a flowing rate of 0.3 mL/min. The injection volume was 5 μL. The electrospray ionization (ESI⁺) MS analysis was performed under positive ion mode with multiple reaction monitoring (MRM) mode. The specificity of each monitoring ion pairs of Cervi Cornu Pantotrichum and Rangifer tarandus Linnaeus were well distinguished and characterized. All 30 batches of Dingkundan samples were detected with components originated from Pantotrichum, 8 batches were detected with components from Cervi Cornu Pantotrichum, and the other 22 batches were detected with ingredients from Rangifer tarandus Linnaeus, indicating more ingredients from pantotrichum in Dingkundan in present markets comes from Rangifer tarandus Linnaeus. The UPLC-MS/MS method has good specificity, features with simple and quick operation, further improving the quality standard of Dingkundan and providing reference for the identification of Cervi Cornu Pantotrichum and screening of Rangifer tarandus Linnaeus in traditional Chinese medicine formulae containing Cervi Cornu Pantotrichum.