Abstract: In order to study polyphenols in tobacco, a method for simultaneous determination of ten endogenous polyphenols in tobacco was developed with high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-TQMS). The ten polyphenols included kaempferol, cyanidin, delphinidin chloride, rhamnazin, chlorogenic acid, delphinidin 3-O-β-glucopyranoside chloride, procyanidine B2, keracyanin chloride, rutin, and scopoletin. The characteristic fragment ion peaks of the ten polyphenols were obtained by dynamic multiple reaction monitoring (DMRM). Tobacco mosaic virus (TMV)-infected tobacco samples were also included in the study. The results showed that:1) The linear correlation coefficients (r) of the ten polyphenols were from 0.999 0 to 0.999 6. The limits of detection (LODs) for all the analytes were 1-200 μg/L, and the intra-day and inter-day relative standard deviations (RSDs) were 0.64%-8.72% and 1.05%-7.93%, respectively. The recoveries were in the range of 79.66%-112.74%. 2) The contents of kaempferol, cyanidin, delphinidin chloride, delphinidin 3-O-β-glucopyranoside chloride and rutin in TMV-infected tobacco samples were significantly higher (p < 0.01) than those in the control, and the rhamnazin content was significantly lower (p < 0.05) and the chlorogenic acid content was higher in TMV-infected tobacco than in the control. 3) The contents of procyanidine B2, keracyanin chloride, and scopoletin were not significantly affected by TMV infection. This method could be used to simultaneously determine a variety of endogenous polyphenols with good sensitivity, precision and recovery.