The co-occurrence of pesticides in aquatic ecosystems highlights the need for studies investigating their potential toxicity as mixtures to the aquatic biota. Well-designed studies are essential to assess the presence and toxicity of relevant pesticide mixtures, particularly those such as the chloroacetamide herbicide metolachlor (MET), the triazole fungicide epoxiconazole (EP) and the diamide anthranilic insecticide chlorantraniliprole (CAP), which have not been previously tested, and whose co-occurrence is possible in waters close to cultivated areas. A solid phase extraction ultra-performance liquid chromatography-tandem quadrupole mass spectrometry method was developed to quantify equivalent toxicity concentrations for CAP, EP, and MET in artificial freshwater during acute toxicity tests. Compounds were separated within 1.30 min, showing linearity over the calibration ranges of 2-150 µgL