Melatonin is an indoleamine, which plays a significant role in regulation of the physiological activities and stresstolerance of plants. This study employed Ultra Performance Liquid Chromatography tandem Mass Spectrometry (UPLCMS/MS) and measured the changes of melatonin content in ‘Cabernet Sauvignon’ grape leaves and roots under the conditions of salt and drought stress, respectively, and explored the physiological functions and regulation mechanisms of melatonin in response to stress. The results showed that: (1) the UPLCMS/MS method for determination of melatonin was optimized, and melatonin was extracted from different grape tissues by the method of ultrasonication. The HPLC condition was as follows: The analytes were separated using an Agilent Eclipse XDBC18（1.8 μm，3.0×50 mm）at 42 ℃ with linear elution gradient at 0.2 mL/min flow rate. The injection volume was 1 μL. Mass spectrometric conditions: The electrospray was operated under the positive ionization mode and the sample was identified by multiple reaction monitoring (MRM) mode. The ion of monitor was at m/z 233→174. The established method exhibited high specificity and sensitivity, the relative standard deviation (RSD) was less than 7%. In addition, the limit of detection (LOD) and the limit of quantity (LOQ) was 0.04 ng/mL and 0.12 ng/mL, respectively. (2) The treatments of drought and salt stress significantly increased the melatonin content in leaves and roots of grape seedlings, and the increase range of melatonin content was enlarged with the increasing degree of stress treatment. After the seedlings were treated with 120 mmol/L NaCl, the melatonin content of grape roots and leaves reached 627.25 and 3 220.42 pg/g, respectively, which was about 7 times higher than that of control seedlings. When treated with 10% PEG6000, the content of melatonin in roots and leaves was much higher than that in the control group. As a conclusion, UPLCMS/MS is an accurate and efficient method for the detection of endogenous melatonin in plants. the melatonin biosynthesis in grape is a response to stress, indicating that melatonin may play a vital role in alleviating abiotic stress in grape tissues.