Free amino acids play an important role in smokeless tobacco taste and cigarette smoke flavor. Amino acid levels change during tobacco aging, curing and tobacco manufacturing process. The objective of this research was to develop and validate a rapid and robust method for the analysis of 17 free amino acids in tobacco products using ultra performance liquid chromatography (UPLC) with ultraviolet (UV) detection. Tobacco samples were extracted with MilliQ water and an aliquot of extract was acidified with 0.1N HCl. The acidified extract was derivatized using Waters AccQ •Fluor Reagent (6-aminoquinoyl-N-hydroxysuccinimidyl carbamate (AQC) and the derivatives were analyzed using UPLC with UV detection. An Acquity UPLC BEH C18 column (1.7 µm particle size) was used and the mobile phases were Waters AccQ•Tag Ultra Eluent A and Eluent B. All requirements for method validation were met including linearity, accuracy, precision, limits of detection (LOD), limits of quantitation (LOQ), method robustness, and standard and sample extract stability. For example, the linearity was demonstrated with a calibration range of 5 to 900 pmol/µL (0.3 to 180 µg/mL) with a coefficient of determination of R2>0.995 for each amino acid. The recoveries for 17 amino acids in tobacco ranged between 90%-112%. The limit of quantitation (LOQ) ranged from 3.0 to 13 µg/g in tobacco for the various amino acids. Once validated, this method was used to evaluate CORESTA reference tobacco products CRP1, CRP2, CRP3, CRP4 and 3R4F tobacco filler where amino acid levels were detected from below LOQ to 6000 µg/g.