To identify the specific biomarkers of exposure of DON in chickens, a toxicokinetic study was performed via oral or intravenous application of deoxynivalenol (DON). Doses of 0.75 and 2.25 mg DON/kg of body weight (BW) were administrated intravenously or orally to the chickens. Next, blood samples were collected at several time points and plasma was obtained. Liquid chromatography–tandem massspectrometry (LC–MS/MS) was used to quantify plasma levels of DON and its metabolite DON-3- sulphate (DON-3S). A non-compartmental analysis was performed to study the main toxicokinetic parameters after intravenous or oral application of the toxin. Regarding oral administration, DON plasma level was below the limit of detection (LOD) of the method (1.5 ng/mL) and DON-3S could not be identified. After intravenous administration of DON at 0.75 and 2.25 mg DON/kg BW, the elimination half-life was 57.1 and 47.7 min, respectively, indicating the rapid elimination of DON. The metabolite DON-3S was found in plasma of broilers exposed to DON intravenously. The absence of DON in chicken plasma after oral bolus application suggests the low absorption of this mycotoxin. The presence of DON-3S in plasma indicates that this metabolite could be the appropriate biomarker of DON exposure in chickens.