The levels of amino acids in tobacco are important for leaf characterization. They contribute substantially to the level of toxicants in cigarette smoke, and to the sensory properties of cigarettes. The analysis of amino acids in tobacco leaf has been practiced using a variety of analytical techniques, but difficulties are still encountered with the analysis. A common procedure uses derivatization with o-phthalaldehyde-fluorenylmethyl chloroformate (OPA-FMOC) followed by HPLC analysis with fluorescence detection. This technique provides reliable results, although has shortcomings: uses derivatization; the freshness of derivatization reagents influences the accuracy or results; amino acids are recognized only based on their retention times in the chromatograms; it is limited in covering a range of amino acids concentrations. An improved original procedure for amino acid analysis has been developed by separating the un-derivatized amino acids by ion-pair reversed-phase HPLC with detection performed using a tandem mass spectrometer. This new procedure resolves the shortcomings of OPA-FMOC analysis and provides accurate and precise results in the analysis of 21 amino acids. The procedure has been used for comparing the amino acid content in 15 different tobaccos. Although based on a limited number of tobacco types, the study provides a comparison of flue-cured, burley, and Oriental tobaccos, leaf + some tips vs. lower stalk, and different growing locations. Besides the expected differences between flue-cured, burley, and Oriental(s), the study showed that the difference in amino acid profile between leaf + some tips vs. lower stalk is mainly of quantitative nature. Also, it showed that the growing location for the tobacco alters the ratios of the levels of different amino acids, but maintains rather similar profiles for the same tobacco type.