Exposure to anthropogenic chemicals poses a global challenge to both environmental systems and human health. Using effect-based human biomonitoring (EB-HBM), we characterize human-relevant chemical mixtures and assess their potential adverse effects. EB-HBM combines the in silico prioritization of target chemicals, exposure assessment using exhaustive extraction and high-resolution mass spectrometry, and the evaluation of biological effects in health-relevant cell-based bioassays. In this seminar, we will demonstrate how we used computational modeling of artificial mixtures to prioritize thousands of chemicals with immunotoxic potential and subsequently tested selected endocrine disruptors as human-relevant mixtures using a comprehensive panel of in vitro bioassays. We will further discuss the importance of considering effect recovery alongside broad chemical coverage when assessing the suitability of extraction methods for human biomonitoring. To conclude, we will demonstrate the implementation of in vitro bioassays within a One Health framework using results which revealed that the effects of complex chemical mixtures propagate across environmental, food, and human samples.