A holistic, simultaneous, and sensitive monitoring of multiple chemical exposures is not yet available, therefore an ultra-sensitive LC-MS/MS method to determine a wide variety of synthetic and natural xenobiotics (>80) in three clinically-relevant human bio-fluids (urine, serum/plasma, breast milk) is developed in this work. To demonstrate the potential of the method for detecting trace-level chemical contamination in precious, low-volume samples (< 100 µL), obtained from a cohort of extremelypremature babies are investigated. This study is an unprecedented assessment of early-life exposure in the blood of a particularly vulnerable patient subgroup. Additionally, longitudinal breast milk samples covering the first 211 days of life from one mother are screened for potentially adverse quantities of xenobiotics and estimations of daily intake are related to current exposure reference values. We show that preterm neonates are exposed to 27 of the included compounds, partly to significantly higher levels than adult controls. Moreover, breast milk acts as a vehicle of 29 included xenobiotics to the infant, however not at critical levels according to current health recommendations. Most importantly, the method demonstrates high sensitivity and selectivity for multilpe chemical classes, as well as an extendable nature. This enables its future application in large-scale cohorts. Schematic illustration depicting the application of a comprehensive exposome approach in an epidemiological setting to improve our understanding of the impact of chemical co-exposure on chronic disease. The prevention of disease will be aided by informed policy decisions and individualized precision medicine.