Using the HPLC/DAD/ESI/MS method, the qualitative and quantitative analysis of senkyunolide A (SA) in the rhizomes of Ligusticum chuanxiong (Rhizoma chuanxiong; CX) and roots of Angelica sinensis (DG) was established. As a result, it was found that SA is a characteristic standard compound for the quality evaluation and chemical differentiation between CX and DG. Methanol was chosen in the preparation of standard solutions and extraction of samples based on the stability data. The identity of SA in CX and DG was unambiguously determined based on the quasimolecular ions in ESI-MS. A comprehensive validation of the method, including sensitivity, linearity, reproducibility and recovery, was conducted using the optimized chromatographic conditions. The linear calibration curve was acquired with R²>0.999 and limit of detection (S/N=3) was estimated to be 12.5 μg/g. The reproducibility was evaluated by repeated sample injection and replicated analysis of samples with the relative standard deviation (RSD) value found within 0.68%. The recovery rates of SA varied within the range of 96.91—101.50% with RSD less than 2.38%. In the present work, the contents of SA were quantified within 3.94—9.14 mg/g and 0.108—0.588 mg/g for 12 batches each of CX and DG. The results demonstrated that SA is a useful standard compound for the quality evaluation and chemical differentiation between CX and DG. The analytical procedure is precise and reproducible and thus suitable for the analysis of a large number of samples.