A fast and efficient multiresidue extraction-purification procedure was developed for low levels (ppb range) of phenanthrene and hydroxyphenanthrene in biological matrices, in order to quantify phenanthrene and metabolites in blood, milk, urine, and biological tissues of lactating goats. Detection and identification of the analytes (phenanthrene and 1-, 2-, 3-, 4-, and 9-hydroxyphenanthrene) were achieved using gas chromatography coupled to mass spectrometry. Deuterium-labeled phenanthrene was used as internal standard for phenanthrene and 2-OHfluorene for metabolites. The developed method includes enzymatic hydrolysis, liquid-liquid extraction, and Envi-Chrom P SPE column purification. Analyses were performed in the selected ion monitoring mode to achieve ad hoc sensitivity in accordance with analyte concentrations in food samples. Detection limits were between 2.3 and 5.1 ng/mL (ppb) for milk samples, 0.5 and 2.5 ng/mL for urine and blood samples, and 1.9 and 8.0 ng/g for tissue samples. This original multiresidue and multimatrix analytical methodology was applied to metabolism studies and polycyclic aromatic hydrocarbon (PAH) risk assessment. We demonstrated, for the first time, that metabolites were present in milk. These results suggested that usual PAHs analysis methods based on the detection of native molecule are not representative of the real contamination of biological matrices.