Analytical methods to quantify pesticide biomarkers in human population studies are critical for exposure assessment given the widespread use of pesticides for pest and weed control and their potential for affecting human health. We developed a method to quantify, in 0.2 mL of urine, concentrations of 10 pesticide biomarkers: four organophosphate insecticide metabolites (3,5,6-trichloro-2-pyridinol (TCPy), 2-isopropyl-6-methyl-4-pyrimidinol, para-nitrophenol, malathion dicarboxylic acid); five synthetic pyrethroid insecticide metabolites (4-fluoro-3-phenoxybenzoic acid, 3-phenoxybenzoic acid, cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid); and the herbicide 2,4-dichlorophenoxyacetic acid. he method is based on enzymatic hydrolysis of conjugated urinary metabolites, extraction and pre-concentration of the deconjugated metabolites using automated online solid-phase extraction, and separation and quantification using liquid chromatography-isotope dilution tandem mass spectrometry. Depending on the analyte, method detection limits were 0.1–0.6 ng/mL; mean accuracy, calculated as spike recoveries, was 91–102%, and total precision, given as percent variation coefficient, was 5.9–11.5%. Percent differences associated with three freeze-thaw cycles, 24-h benchtop storage, and short-term processed sample stability were <14%. Method suitability was assessed by recurring successful participation in external quality assessment schemes and by analyzing samples from subjects with suspected exposure to pesticides (n = 40) or who self-reported consuming an organic diet (n = 50). Interquartile ranges were considerably lower for people consuming an organic diet than for those potentially exposed for cis-DCCA (0.37 ng/mL vs 0.75 ng/mL), trans-DCCA (0.88 ng/mL vs 1.78 ng/mL) and TCPy (1.81 ng/mL vs 2.48 ng/mL). This method requires one-fifth of the sample used in our previous method and is suitable for assessing background exposures to select pesticides in large human populations and for studies with limited sample volumes. Display omitted •Novel LC-MS/MS method for quantifying pesticide biomarkers in urine•Online sample cleanup and isotope dilution LC-MS/MS pesticide exposure method•Method uses 0.2 mL urine and is sensitive, reproducible, and accurate•Suitable to assess background exposures in large-scale population studies