Abstract: To establish a scientific and objective method for assessing the roasting uniformity of burley tobacco, four Maillard reaction intermediates (glucosamine, fructose-glutamic acid, fructose-phenylalanine and fructose-asparagine) were selected as the characteristic markers. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantitative analysis of the four markers from the roasted tobacco leaves. The sampling protocol was optimized using multivariate statistical analysis, and then a multi-indicator assessment strategy for the roasting uniformity of burley tobacco was established by analyzing the Maillard reaction intermediates. The results showed that: 1) The selected markers showed satisfactory applicability for characterizing roasting uniformity. The sampling protocol of 50 g sample mass, 30 sampling repetitions, and 60 s sampling intervals showed better representativeness and could meet the analytical requirements. 2) The method showed robust stability with the coefficients of variation (CV) ranging from 2.5% to 3.3% for the contents of the four Maillard reaction intermediates in blended burley tobacco leaves over different production batches of the same specification. The coefficients of roasting uniformity reached 91.5%-93.2% with a batch-to-batch CV of 0.9%. In conclusion, the established approach to assess roasting uniformity was stable and the assessment results for the roasting uniformity of burley tobacco from different production batches of the same specification were consistent. Compared with the traditional moisture content detection, the established method objectively assessed the intrinsic quality consistency of burley tobacco roasting processes by quantifying the Maillard reaction product distribution, and provided a novel tool for optimizing the blended cigarette manufacturing process and identifying potential process deficiencies.