Breast cancer (BC) is one of the most common malignant tumors among women worldwide and can be treated using various methods; however, side effects of these treatments cannot be ignored. Increasing evidence indicates that compound kushen injection (CKI) can be used to treat BC. However, traditional Chinese medicine (TCM) is characterized by "multi-components" and "multi-targets", which make it challenging to clarify the potential therapeutic mechanisms of CKI on BC. Herein, we designed a novel system pharmacology strategy using differentially expressed gene analysis, pharmacokinetics synthesis screening, target identification, network analysis, and docking validation to construct the synergy contribution degree (SCD) and therapeutic response index (TRI) model to capture the critical components responding to synergistic mechanisms of CKI in BC. Through our designed mathematical models, we defined 24 components as a high contribution group of synergistic components (HCGSC) from 113 potentially active components of CKI based on ADME parameters. Pathway enrichment analysis of HCGSC targets indicated that Rhizoma Heterosmilacis and Radix Sophorae Flavescentis could synergistically target the PI3K-Akt signaling pathway and the cAMP signaling pathway to treat BC. Additionally, TRI analysis showed that the average affinity of HCGSC and targets involved in the key pathways reached -6.47 kcal/mmol, while in vitro experiments proved that two of the three high TRI-scored components in the HCGSC showed significant inhibitory effects on breast cancer cell proliferation and migration. These results demonstrate the accuracy and reliability of the proposed strategy.Copyright © 2021 Li, Wang, Chen, Cai, Qin, Lu, Guan, Qin and Chen.