Abstract: In this study, a rapid and sensitive method based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was established for the trace determination of benzohydroxamic acid and applied to the quantitative analysis of benzohydroxamic acid in wheat plants and nutrient solutions under hydroponic exposure conditions. The results showed that benzohydroxamic acid exhibited good linearity over the concentration range of 10-5000 μg/L in wheat plants and nutrient solutions, with correlation coefficients (R²) ≥ 0.9919. The limit of quantification (LOQ) was 10 μg/L, the matrix effect ranged from −9.5% to −3.1%, the average recoveries were 70%-94%, and the relative standard deviations (RSDs) were 4.1%-16.2%, indicating that the method met the requirements for trace quantitative analysis of benzohydroxamic acid in different wheat matrices, including wheat roots, shoots, and nutrient solution. Further hydroponic exposure experiments showed that benzohydroxamic acid could be rapidly taken up by wheat roots and translocated to shoots. The uptake processes in both roots and shoots could be divided into a rapid uptake phase (0.125-3 d) and an accumulation equilibrium phase (3-21 d), overall exhibiting kinetic characteristics of rapid uptake, accumulation equilibrium, and relatively fast elimination, with roots serving as the primary accumulation site. This method provides basis dates for investigating the uptake and translocation of benzohydroxamic acid in wheat and supports its subsequent application safety evaluation.