Analytical methods have been developed for the detection and quantitation of a new herbicide active ingredient, aminocyclopyrachlor, and its analogue aminocyclopyrachlor methyl in environmental samples. The analytes were purified from soil extracts and water samples using solid phase extraction based on mixed-mode cation exchange/reverse phase retention. Analyte identification and quantitative analyses were performed by high performance liquid chromatography coupled to tandem mass spectrometry by an electrospray ionization source. External standards prepared in neat solvents were used for quantitation, providing acceptable accuracy, with no matrix effects observed during method validation. The method limits of quantitation (LOQ) were 0.10 ng/mL (ppb, parts-per-billion) in water and 1.0 ng/g in soil for both compounds. The limit of detection (LOD) in water was estimated to be 20 ng/L (ppt, parts-per-trillion) for aminocyclopyrachlor and 1 ng/L for aminocyclopyrachlor methyl, while LODs in soil were 100 ng/kg and 10 ng/kg for aminocyclopyrachlor and aminocyclopyrachlor methyl, respectively. The stability of both compounds in various solvents was evaluated as part of method development. Tandem mass spectrometry experiments were also conducted to investigate the gas-phase fragmentation of aminocyclopyrachlor and its methyl analogue, and the results are reported. A statistical analysis of method validation data generated at two laboratories by multiple chemists authenticates the ruggedness and good reproducibility of the analytical procedures tested.