This publication reports the first known use of gas chromatography-tandem mass spectrometry for the quantitation of five minor tobacco alkaloids (nornicotine, myosmine, anabasine, anatabine, and isonicoteine) in various tobacco samples. A summary of the concentrations of these minor alkaloid levels in the filler from 50 popular cigarette brands were found to be 659-986 μg/g nornicotine, 8.64-17.3 μg/g myosmine, 127-185 μg/g anabasine, 927-1390 μg/g anatabine, and 23.4-45.5 μg/g isonicoteine. Levels of minor alkaloids found in reference cigarettes (1R5F, 2R4F, 3R4F, CM4, and CM6) as well as burley, flue-cured, oriental, reconstituted, and Nicotiana rustica and Nicotiana glauca tobacco types are also reported. Quantitation of the minor tobacco alkaloids is important because the alkaloids have been shown to be precursors of carcinogenic tobacco specific N'-nitrosamines.