A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC-MS/MS) and high-resolution Orbitrap(®) mass spectrometry ((U)HPLC-HR-MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 μm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases. Both mass spectrometers were operated in the negative h-ESI mode. The method was in-house validated for all analytes: matrix-matched calibration graphs were prepared and good linearity (r≥0.99) was achieved over the concentration range tested (0.2-200 ng mL(-1)). Limits of quantification (LOQ) in plasma were between 0.2 and 5 ng mL(-1) for all compounds. Limits of detection in plasma ranged from 0.004 to 0.070 ng mL(-1). The results for the within-day and between-day precision, expressed as relative standard deviation (RSD), fell within the maximal RSD values (within-day precision: RSD(max)=2((1-0.5logConc)) x 2/3; between-day precision: RSD(max)=2((1-0.5logConc))). The accuracy fell within -50% to +20% (concentrations <1 ng mL(-1)), -30% to +10% (concentrations between 1 and 10 ng mL(-1)) or -20% to +10% (concentrations >10 ng mL(-1)) of the theoretical concentration. The method has been successfully used for the quantitative determination of ZEN in plasma samples from broiler chickens and pigs. α-ZEL and β-ZEL were the only metabolites that could be detected, but the concentrations were around the LOQ levels. The intact ZEN-glucuronide conjugate could be detected using the (U)HPLC-HR-MS instrument. A good correlation (r(2)=0.9979) was observed between the results for ZEN obtained with the LC-MS/MS and (U)HPLC-HR-MS instruments. The results prove the usefulness of the developed method for application in the field of toxicokinetic analysis and for exposure assessment of mycotoxins.