A rapid analytical procedure for the simultaneous determination of twenty seven hormones in fish, milk and water samples by solid-phase extraction (SPE) and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed.These hormones, considered as endocrine-disrupting chemicals (EDCs), contained nineteen endogenous and eight exogenous.The method involves extraction with acetonitrile and purification by a HLB SPE cartridge.In order to optimize purification effect of milk and fish matrixes, a comparative study using a diphasic dialysis and matrix solid-phase dispersion (MSPD), was weak in achieving the maximum retention of analytes and best elimination of matrix components.Using the optimized protocol of a HLB cartridge, limits of quantification (LOQs) were obtained ranging from 0.1 to 49.5 ng g-1 for water samples, from 0.1 to 39.1 ng g-1 for milk samples and from 0.1to 49.5 ng g-1 for zebrafish (Danio rerio) samples.Spiked recoveries were within 69.1-120.5% of nominal concentrations.Performance characteristics permit the use of this methodology for 27 hormones determination in fish, milk, water samples.Hydrocortisone (Hd), androstenedione (AN), stanozolol (STD), progesterone (P4), 17-hydroxyprogesterone (17-OHP) and 17-β-Estradiol (E2) were found in milk ranging from 0.21 to 11.41 ng g-1.P4 and trenbolone (TB) were observed in zebrafish at 5.73 and 7.45 ng g-1, respectively.This multi-residue method was used for quantitation of hormone levels in a model organism, zebrafish, tested in endocrine disruptor screening experiments.Meanwhile, this technology would play an important role in evaluating the presence of target EDCs in food and environmental samples.