Abstract: To develop a rapid method for simultaneous determining 16 free amino acids and 6 Amadori products (APs) in tobacco, a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. After extracting with water, tobacco samples were separated on an Acclaim Explosive E2 column with the gradient elution mobile phase of methanol and 0.1%(V/V) formic acid aqueous solution. The mass spectrometry detection was carried out by means of electrospray ionization in positive mode (ESI⁺) with multiple reaction monitoring(MRM)method. The results showed that:1)The correlation coefficients(r²) were higher than 0.996 for the all calibration curves, the standard recoveries of the spiked samples ranged from 82.0% to 109.5%, the intra-day and the inter-day relative standard deviations(RSDs)were 0.6%-4.8%(n=6) and 1.3%-10.9% (n=6), respectively. 2) This method was simple, rapid, accurate, and suitable for the simultaneous determination of the free amino acids and APs in tobacco samples.