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Butachlor is an aromatic amide compound that plays a role as a herbicide, a xenobiotic, and an environmental contaminant. The aim of this work was to develop a highly selective and sensitive ultra‐performance liquid chromatography‐tandem mass spectrometry method based on the tandem mass spectrometry cubed technique to determine butachlor in a biological matrix. Butachlor and internal standard acetochlor were separated on a Waters Acquity ultra‐performance liquid chromatography BEH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution using 0.1% formic acid aqueous solution (A) and acetonitrile (B) as mobile phases. The transitions selected for tandem mass spectrometry cubed quantitative analysis in positive ion mode were: for butachlor, mass‐to‐charge ratio 312.2→238.1→162.1; for acetochlor, mass‐to‐charge ratio 270.1→224.0→148.1. The total running time for each sample was 5.5 min. The ultra‐performance liquid chromatography‐tandem mass spectrometry cubed method showed a linear relationship (R2 ≥ 0.995) in the concentration range of 0.5–100 ng/ml. The intra and interday accuracies are within the range of ‐10.6%–4.3% and precisions are between 4.48% and 13.14%. The novelty of the method is the use of tandem mass spectrometry cubed scanning mode, which improves selectivity and sensitivity. The results indicated that butachlor was cellular toxic. The safety of butachlor should be considered when it is used as a herbicide.

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