The optimized LC-MS/MS method was thoroughly evaluated by analytical validation according to the guidelines established by the European Commission Decision N o. 657/2002 , with minor modifications. Urine, serum, and breast milk samples were chosen for validation and included extraction experiments at two trace-level concentrations. Overall, 75% and 79% of the 81 analytes fulfilled all criteria for at least one concentration level in urine and serum, respectively. For the more-challenging breast milk matrix, the percentage was lower at 56%. For seven analytes, the selectivity, recovery, or linearity were outside the guideline ranges in all matrices. Although these analytes are restricted to semi-quantitative or qualitative evaluation, the information obtained can still be valuable in environmental-health studies. Importantly, validation guidelines are still focused on tailored, single analyte assays and typically do not encourage multi analyte assays. As differing sensitivities between bio-fluids were expected in our novel next-generation HBM method, it was anticipated that not all compounds would completely fulfil the criteria at all chosen fortification levels in every matrix. Particularly, since fortification and calibration were based on the most sensitive matrix for each analyte (see Supplementary Table ).