Chuanxiong Chatiao powder is a classic prescription in traditional Chinese medicine. A variety of dosage forms have been developed based on powders, such as dropping pills, granules, tablets, pills, and concentrated pills. This classic prescription consists of various active constituents, but due to the selection of a single and unreasonable quality standard indicator, may cause a difference in active constituent and bioactivity among different dosage forms. However, related reports on the comparisons of the different dosage forms are few. Therefore, high-performance liquid chromatography with triple quadrupole mass spectrometry was developed for the determination of 26 bioactive components of the six Chuanxiong Chatiao dosage forms, and a parallel line assay based on a quantitative response method for biological assays was established to measure the antiplatelet aggregation biopotency (AAB) in vitro. The results indicated that the AAB of the powder was greatest, followed by that of the pills, concentrated pills, dropping pills, tablets, granules. The results were analyzed by hierarchical clustering analysis, showing that were some differences among six dosage forms. 12 different markers, including Z- ligustilide, senkyunolide A, butylidenephthalide, asarinin, rosmarinic acid, pulegone, senkyunolide H, notopterol, coniferyl ferulate, hesperidin, vanillic acid, luteolin-7-O-glucoside, were further screened using partial least squares. All of different markers showed a positive association with AAB. There were significant differences in both the sum and composition of different markers among the different dosage forms. This can provide a foundation for the clinical study of the six Chuanxiong Chatiao dosage forms.