A simple and sensitive method for the separation and determination of zearalenone (ZON) and its secondary metabolites, namely beta-zearalenol (beta-ZAL), beta-zearalanol (beta-ZOL), alpha-zearalenol (alpha-ZAL), alpha-zearalanol (alpha-ZOL) and zearalanone (ZAN) in urine (human, bovine and swine) samples is proposed. The method comprises previous clean-up and pre-concentration on C(18) adsorption cartridges of the analytes followed by liquid chromatographic separation and direct electrochemical detection at +0.85mV using a carbon nanotube-modified glassy carbon electrode (CNT-GCE). This method allows the determination of beta-ZAL, beta-ZOL, alpha-ZAL, alpha-ZOL, ZAN and ZON in a linear range between 5 and 50ngmL(-1), with relative standard deviation values lower than 6.9% (intra-day) and 7.1% (inter-day), in all cases. Detection limits ranging between 1.3ngmL(-1) (beta-ZOL, alpha-ZAL, alpha-ZOL) and 1.4ngmL(-1) (beta-ZAL, ZAN, ZON) were achieved. The usefulness of the proposed method was demonstrated by the analysis of spiked and natural samples of human, bovine and swine urine samples.