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The increasing number of children suffering from developmental disorders has raised questions regarding their association with the presence of environmental contaminants in mothers and children. We therefore developed a new method for the determination of 78 proven and potential developmental neurotoxicants, including polychlorinated biphenyls, legacy pesticides, pyrethroids, and old and new halogenated flame retardants in breast milk. The essential part of sample preparation was dialysis as a non-destructive clean-up step which was newly used at 10 °C and showed more efficient lipid removal (up to 96%) than the conventional methods such as gel permeation chromatography or freezing-lipid filtration and thus ensured low limits of detection (LOD) by reducing the sample volume prior to injection. Next advantages were significant solvent reduction and no risk of sample cross-contamination. Gas chromatography coupled with high resolution mass spectrometry (GC-HRMS) was subsequently used for the separation and compound quantification. The method was validated using breast milk samples fortified with the analyzed compounds. Recoveries for most of the compounds ranged from 63 to 121% with a relative standard deviation of 2-25%, and LODs ranged between 0.001 and 0.87 ng g lipid weight. The method was applied to breast milk samples from a Dutch birth cohort where 35 out of the 78 compounds were quantified in more than 60% of the samples. For novel flame retardants, the method provides unique results regarding their occurrence in human matrices in Europe. Overall, the analysis of a complex mixture of developmental neurotoxicants could be useful for the assessment of the influence of the studied compounds to child health and development. Graphical abstract Flow diagram of the method and levels of the developmental neurotoxicants in Dutch human milk samples.