Abstract A sensitive gas chromatographic (GC)—ion-trap mass spectrometric (IT-MS) method has been developed to determine simultaneously the herbicides glyphosate, glufosinate and bialaphos and their major metabolises. A single-step derivatization is achieved at 80°C for 30 min with the reagent N-methyl-N-( tert.-butyldimethylsilyl)trifluoroacetamide in dimethylformamide, which introduces the tert.-butyldimethylsilyl group at active hydrogens and gives only a single peak for each compound. The derivatives of three herbicides, their metabolises and nineteen amino acids were simultaneously chromatographed and well separated in a single run on a DB-1 fused-silica capillary column. Each tert.-butyldimethylsilylated derivative produces an easily interpretable mass spectrum dominated by unique M - 15, M - 57, M - 85 and M - 159 fragment ions. The limits of detection were estimated to be 10–20 ng for glyphosate and glufosinate and their metabolises, and 500 ng for bialaphos, by GC-IT-MS. On the other hand, using GC analysis with flame ionization detection, glyphosate and glufosinate and their metabolises were detectable at levels of approximately 100 ng, but bialaphos could not be detected at a level of 5000 ng.