A simple and sensitive automatic amino acid analyzer method for the determination of free amino acids in tobacco was described. Sample preparation consisted of a single step of extraction with 0.1 mol HCl at ambient temperature in 60 min by sonication, followed by filtration of an aliquot. Automated amino acid analyzer was used to construct a post-column ninhydrin reaction unit to monitor amino acids separated by liquid chromatography using a series of eluting buffers. By optimization of sample preparation, separation of 19 amino acids was achieved. Limits of quantitation was 0.01 mg/g, coefficients of variation ranged from 0.5 % to 8.9 % and recoveries range from 85 % to 106 %. The method was applied to the analysis of amino acids contents of tobacco leaves in different varieties.