An automated online solid-phase extraction–liquid chromatography mass spectrometry (SPE–LC/MS) method was developed for the quantification of dithianon in surface water samples, using warfarin as internal standard. The method was developed on a liquid chromatography (LC) system with Flexible Cube interfaced to a quadrupole time-of-flight (Q-TOF) mass spectrometer. A small volume of acidified water (1 mL) was spiked with internal standard, pre-concentrated online on polymeric cartridges and analyzed by full-scan MS in high-resolution conditions. The quantitative data were obtained by [M]^−• of dithianon and [M – H]⁻ of warfarin, used as internal standard. The chromatographic separation was performed on a C₁₈ column with a gradient mobile phase consisting of acetonitrile and water containing 0.05% acetic acid. The method was validated to measure concentrations of dithianon in the range of 0.010–4 μg L⁻¹ in surface water samples. Twenty real water samples, collected from Torrente Novella, Val di Non (TN, Northern Italy), during fungicide treatments of large apple orchards, were analyzed. All samples were kept in glass bottles and stored in the lab at −20°C until analysis. It was found that in all samples dithianon was undetectable: if it is present, its concentration was lower than the limit of detection (LOD) (0.008 μg L⁻¹). To investigate the stability of dithianon, a series of water samples were spiked at different concentrations and analyzed after different storage conditions. Results suggested that dithianon is not stable in water stored at −20°C at neutral or basic pH, but the addition of acetic acid to pH = 3.5 increases its stability to at least two weeks.