Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE) treatment. An ultra-performance liquid chromatography (UPLC) method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS) LC−MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2 -Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 µg·g −1 ) at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-D-glucopyranoside. The content of 2 -Hydroxyaucuparin reached its highest (422.75 µg·g −1 ) at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.