Humans can be exposed to mycotoxins through the diet. Evaluation of exposure levels to mycotoxins can be performed by direct determination in urine. The present work proposes a sensitive ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the determination of zearalenone (ZON) and its five metabolites (α-zearalenol [α-ZOL], β-zearalenol [β-ZOL], α-zearalanol [zeranol, α-ZAL], β-zearalanol [teranol, β-ZAL] and zearalanone [ZAN]) in human urine samples. The method involves the enzymatic hydrolysis of the samples, extraction of the analytes using liquid–liquid extraction (LLE) with ethyl acetate/formic acid (99:1 v/v) and a cleanup step using hexane, prior to their quantification by UHPLC–MS/MS, using an electrospray ionization (ESI) interface in the negative mode. Zearalenone-d 6 (ZON-d 6 ) was used as surrogate. The limits of detection and the limits of quantification ranged from 0.03 to 0.3ngmL −1 and from 0.1 to 1.0ngmL −1 , respectively. The method was validated using matrix-matched calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 96% to 104%, with relative standard deviations lower than 8.5%. This method was satisfactorily applied to 42 urine samples from Tunisian women for the determination of zearalenone and its five metabolites.