Lignans have gained nutritional interest due to their promising role in prevention of life style diseases. However, epidemiological studies are in need of more evidence to link the intake of lignans to this promising role. In this context, it is necessary to study large population groups to obtain sufficient statistical power. Therefore there is a demand for fast, sensitive and accurate methods for quantitation with high throughput of samples. Here we present a validated LC-MS/MS method for quantitation of eight plant lignans (mataresinol, hydroxymataresinol, secoisolariciresinol, lariciresinol, isolariciresinol, syringaresinol, medioresinol and pinoresinol) and the two enterolignans (enterodiol and enterolactone) in both human and pig plasma and urine. The method showed high selectivity and sensitivity allowing quantitation of lignans in the range of 0.024-100 ng/mL and with a runtime of only 4.8 min per sample. The method was successfully applied to quantitate lignans in biofluids from on-going studies with humans and pigs.