A multi-residue analytical methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in aqueous environmental matrices, using nigericin as internal standard. The samples were filtrated prior to solid phase extraction. All compounds were measured using liquid chromatography coupled to tandem mass spectrometry applying electro spray ionisation. The absolute recoveries ranged from 92 to 110% (relative standard deviation: 2–14%) for spiked river water. The final method allowed fordetection of ionophores down to a few ng/L in natural water bodies with LOQs for the entire methodology being 40, 49, 67, and 14 ng/L for lasalocid, monensin, salinomycin, and narasin, respectively.