Abstract: In order to determine the contents of endogenous phytohormones in tobacco and analyze the roles of phytohormones in the resistance, growth and development of tobacco, a method for simultaneously determining the contents of 7 endogenous phytohormones, including 3-indoleacetic acid (IAA), 3-indolebutytic acid (IBA), salicylic acid (SA), gibberellin A3 (GA), jasmonic acid (JA), abscisic acid (ABA) and brassinosteroids (BR) in tobacco was developed by high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The TMV (tobacco mosaic virus)-infected tobacco samples were determined by obtaining the characteristic fragment ion peaks of the 7 endogenous phytohormones under multiple reaction monitoring mode. The results showed that:1) The correlation coefficients (r) of the 7 phytohormones were from 0.998 6 to 0.999 5 in the linear ranges. The limits of quantification (LOQ) for all analytes were between 0.07 and 3.00 μg/L, and the intra-and inter-day relative standard deviations (RSDs) were 0.44%-9.76% for all analytes. The recoveries were in the range of 77.82%-117.63%. 2) The contents of SA, IBA, JA, ABA in TMV-infected tobacco were significantly higher (p < 0.01) than the control sample, while which was significantly lower (p < 0.05) for GA. However the content of BR was basically unchanged. With good sensitivity, precision and recovery, this method is suitable for determining various endogenous phytohormones.