BACKGROUND AND AIM: Epidemiological studies linking insecticide exposure to childhood neurodevelopment have been gaining global attention. Despite the rapid development of the central nervous system in early childhood, studies regarding the biological monitoring of insecticide exposure in diapered children are limited. In this study, we aimed to clarify the concentrations of organophosphate (OP) insecticide metabolites in toddler urine extracted from disposable diapers in Japan. METHODS: We recruited diapered children from the Aichi regional subcohort participants of the Japan Environment and Children's Study (JECS) at the time of their 18-month checkup. A total of 116 children wore designated disposable diapers overnight, which were then sent as refrigerated cargo. The urine was extracted from the diapers using acetone and analyzed by ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) to determine the concentrations of six dialkyl phosphates (DAPs) (i.e., dimethyl phosphate [DMP], dimethyl thiophosphate [DMTP], dimethyl dithiophosphate [DMDTP], diethyl phosphate [DEP], diethyl thiophosphate [DETP], and diethyl dithiophosphate [DEDTP]). DAP absorption into the diapers was quantified to calculate the urinary DAP concentrations. RESULTS: The DAP recovery using the developed method yielded between 54.2% (DEDTP) and 101.4% (DEP). Within-run precision expressed as the relative standard deviation was between 2.4% and 14.7%, and the between-run precision was between 3.1% and 8.5%. A Bland-Altman analysis confirmed the agreement between the results obtained by the developed method and by the measurements for the corresponding urine without diaper absorption. The geometric means (GM) of urinary DMP, DMTP, DMDTP, DEP, DETP, and total DAPs (ΣDAP) were 3.6, 3.9, 0.9, 6.0, 0.6μg/L, and 137.6 nmol/L, respectively. The GM of DEDTP was not calculated due to its low detection rate. CONCLUSIONS: We successfully established a method to measure the DAP concentrations in urine extracted from diapers and this is the first report of these pesticide concentrations in diapered children in Japan.