A comprehensive and generic LC-MS/MS assay to simultaneously identify and quantitate multiple classes of endocrine-disrupting and other potentially-harmful toxicants and contaminants (81 xenobiotics in total) was developed and the performance of the assay critically evaluated. Alongside the assessment of the method's linearity, sensitivity and selectivity, two trace concentration levels were applied in spiking experiments to determine analyte recoveries. The method yielded highly convincing results during validation for urine and serum, resulting in more than 75% of the chemicals included fulfilling all criteria for at least one of the fortification levels. This was despite the use of low sample volumes and the application of stringent guidelines intended for targeted single-analyte assays. Even for breast milk, a complex, high-fat matrix, the results were acceptable (56%). Importantly, compounds that did not pass all criteria may still be assessed in a semi-quantitative manner to potentially draw initial conclusions in future ExWAS.