The herbicide, methyl 2-4-(2,4-dichlorophenoxy)phenoxy proprionate (diclofop-methyl), and three of its degradation products are identified in biofilm using tandem mass spectrometry (MS/MS). Sub-samples of a biofilm cultured in a flow-through system, with the herbicide diclofop-methyl in the nutritive solution, were analyzed using two ion-trap mass spectrometers and the data were compared with those obtained using a hybrid instrument of EBEQ geometry. In order to achieve high throughput, the samples were introduced directly into the ion sources with no further sample preparation. Confirmations were based on data taken for authentic standards. The results indicate that data comparable to those obtained using the hybrid instrument could be achieved using ion-traps, despite the complex nature of the biofilm. This is true both of mass spectra and of product ion MS/MS spectra which were essential for identification of the individual compounds in these complex mixtures. For high-throughput analyses (2–50 runs daily), the quality of the MS/MS characterization was dependent on the background pressure and the source temperature. © 1997 John Wiley & Sons, Ltd.