A method based on solid-phase microextraction (SPME) coupled with GC and ion trap tandem mass spectrometry has been developed for the analysis of nine herbicides and degradation products, among the most frequently found in natural water. A polydimethylsiloxane–divinylbenzene (PDMS–DVB)-coated fiber was selected to extract the analytes directly from the samples over the 0.01–1 μg L−1 concentration range. Optimization of manual and automated SPME was performed on the basis of desorbed amounts, via various factorial experiment designs. Of the two modes, the automated one was found to be the most efficient. Memory effect was avoided owing to the 10-min fiber desorption time. Limits of detection reached down to below 0.01 μg L−1 and repeatability ranged from 3 to 15% in natural water. A validation study was conducted involving the quantitation of the target compounds in Seine water with SPME/GC–MS-MS external calibration.