The application of gas chromatography for the separation of TMS-amino acids from a botanical source was demonstrated. The trimethyl-silyl derivatives of the extracts from germ free tobacco tissue cultures were prepared by reacting amino acid extracts with bis(trimethylsilyl)-trifluoroacetamide (BSTFA) using acetonitrile as a reaction solvent following preliminary separation of the free acids by ion exchange chromatography. Gas chromatographic separation was accomplished with a 10% OV-11 glass column and temperature programming. The findings compare favorably with other chromatographic methods. Structures of the TMS-amino acids were confirmed by gas chromatography-mass spectrometry combination. Mass spectral data for each derivative is presented for the principal protein amino acids observed as well as γ-aminobutyric acid, asparagine, α-aminobutyric acid and β-alanine.