Researchers, product registration personnel, and growers desire the ability to chemically detect residual amounts of herbicides in soil at concentrations below those necessary to cause phytotoxicity to sensitive nontarget or rotational crop plants. Alfalfa, cotton, soybean, and sunflower, crops sensitive to low concentrations of aminocyclopyrachlor in soil, were planted at field test sites approximately 1 yr after aminocyclopyrachlor methyl was applied. Soil samples were collected when rotational crops were planted and were analyzed for aminocyclopyrachlor by a method based on high performance liquid chromatography tandem mass spectrometry (HPLC/MS/MS), with a limit of detection (LOD) of 0.1 part per billion (ppb) (soil oven-dry weight basis). Loglogistic dose–response analysis correlated visual phytotoxic plant responses to residual concentrations of aminocyclopyrachlor in the soil. Concentrations of aminocyclopyrachlor estimated to cause 25% phytotoxicity to alfalfa, cotton, soybean, and sunflower were 5.4, 3.2, 2.0, and 6.2 ppb, respectively, 20 to 60 times greater than the LOD of the analytical method available for soil analysis. Results from these studies suggest this HPLC/MS/MS method of analysis can be used to indicate potential risk and severity of plant response for alfalfa, cotton, soybean, and sunflower, and for other plant species once dose–response curves for these additional species are established. This chemical assay may be particularly important if researchers desire to study the concentration, movement, and dissipation of aminocyclopyrachlor in soil or as part of a forensic investigation to better understand the cause of an unanticipated or undesirable plant response.