This study aims to develop, evaluate, and optimize the application of nearinfrared spectroscopy (NIR) autosampler device equipped with a fiber-optic probe associated with chemometric methods for the fast quantification of routine chemical constituents such as total alkaloids, reducing sugars, nitrate, and ammonia in tobacco. For this purpose, an NIR autosampler device with capacity for 40 samples was used. The spectra were collected in the range of 950 to 2,500 nm, with three spins on the sample, a 3.0-cm 2 sample scanning area, and 2.0 ± 0.2 mm distance between the fiber-optic probe and the sample. Four partial least-squares (PLS) models were developed, and different preprocessing methods were investigated. The predicted results were compared with those obtained from the reference method (continuous-flow analysis), and root mean square error of prediction values of 0.31%, 1.27%, 0.47%, and 0.026% were obtained for total alkaloids, reducing sugars, nitrate, and ammonia, respectively. The proposed method performed well for the analysis of total alkaloids and reducing sugars with an appropriate goodness-of-fit and fair precision. In conclusion, considering the performance of the regression models and the associated environmental and economic advantages, the application of NIR spectrometer autosampler device equipped with a fiber-optic probe associated with a PLS and synergy interval PLS algorithms cannot replace the reference method, but it is a promising tool for tobacco monitoring. K E Y W O R D S chemometrics, fiber-optic probe, NIR autosampler, tobacco 1 | INTRODUCTION Tobacco quality is influenced by the tobacco type, environmental conditions, agricultural practices, and leaf ripeness. During their growth and curing processes, tobacco plants undergo into a series of physiological and metabolic processes. Change in the metabolites can modify the leaf characteristics and consequently affect the chemical composition and organoleptic characteristics in tobacco smoke. 1-3 There are several types of tobacco such as flue-cured Virginia, 1,2 air-cured Burley, 1,2 sun-cured Oriental, 1 air-cured Maryland, 1 and air-cured Dark. 4 Among these, the main ones produced in Brazil are flue-cured Virginia and air-cured Burley. 5,6 Alkaloids, sugars, reducing sugars, nitrate, and nitrogen