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Calculus Bovis (C. bovis) is a precious traditional Chinese medicine of animal origin, and it is one of the traditional medicines for treating cerebral inflammatory diseases such as stroke. However, the pharmacological action of C. bovis on ischemic stroke (IS) and its mechanism are still unclear. The purpose of this study was to investigate the potential mechanism to treat IS. Chemical constituents of different varieties of C. bovis were analyzed and confirmed by HPLC-MS/MS technique. We constructed a component and corresponding target network and drug-disease target protein-protein interaction (PPI) network. GO and KEGG enrichment analysis were performed. The molecular docking of the main compound with the target protein. Subsequently, the potential mechanism of therapy for IS was verified in vivo by zebrafish model. We introduced Raman spectroscopy to detect changes in the biochemical composition of zebrafish. 13 active chemical constituents and 129 potential targets were selected. 122 KEGG signaling pathways were obtained. The binding energy of the main compounds is less than -4.5. The results of animal experiments showed that C. bovis could significantly improve Ponatinib-induced IS, decrease the aggregation degree of brain macrophages, reduce the number of macrophage migrations, and significantly increase the expression level of NR3C1. Raman information indicated that the biochemical composition in the brain of the Ponatinib-induced group shifted to the control group. The mechanism may be related to anti-inflammatory process and regulation of lipid metabolism. This study demonstrates that Raman spectroscopy has great potential as a drug evaluation tool in living larval zebrafish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)