Abstract: Using the in-house synthesized N-(1-deoxy-D-fructos-1-yl)-L-proline(Fru-Pro) and N-(1-deoxy-D-fructos-1-yl)-L-alanine(Fru-Ala) as standards,a HPLC/MS method for qualitatively and quantitatively analyzing these two compounds in tobacco was developed.Sixteen tobacco samples were analyzed,the results showed that:1) Fru-Pro and Fru-Ala were identifiable via retention time and main mass fragmentation under SCAN mode and quantifiable under selected positive ion mode at(m/z)278.0 and(m/z)252.0,respectively;2) The recoveries of Fru-Pro and Fru-Ala were 93.1% and 105.8% with the relative standard deviations of 4.23% and 3.97% and the limits of detection of 25.0 and 30.0 μg/g,respectively;3) The contents of Fru-Pro and Fru-Ala in 3 flue-cured tobacco leaf samples were much higher than that in 3 burley tobacco samples,in 5 domestic Virginian-type cigarette samples were higher than that in 1 imported Virginian-type cigarette sample,2 domestic and 2 imported blended-type cigarette samples.The method was suitable for the determination of these two compounds in tobacco samples.