A method that measures 12 analytes in urine and reflects possible exposure to pesticides was developed. The sample preparation involves enzyme hydrolysis and solvent extraction through the use of laboratory robotics, followed by phase-transfer catalysis derivatization and silica cleanup. Samples are analyzed by capillary gas chromatography and tandem mass spectrometry using an isotope dilution technique with 13C-labeled internal standards. The limit of detection is 1 microgram/L (1 part per billion) for most analytes, and most analytes have a linear response up to 100 micrograms/L. The precision of the method is reflected in the variation observed in quality control materials over 33 months; the variation averaged 17% for these analytes. On the basis of the detectable analyte levels of unspiked urine samples collected from unexposed volunteers, this method can be used to measure the low levels necessary for establishing reference range values of the selected pesticides or metabolites.