Abstract: This study aimed to determine the conditions for establishing a zebrafish migraine model and to evaluate the therapeutic effects of Tianshu Capsules against migraine. A behavioral monitoring system was used to assess changes in zebrafish locomotor activity following nitroglycerin injection. Dopamine (DA) concentrations in zebrafish brain tissues were measured using an enzyme-linked immunosorbent assay (ELISA), and the mRNA expression levels of il-1b, nos2b, c-fos, and c-jun were determined by quantitative polymerase chain reaction (qPCR). Hematoxylin and eosin (HE) staining was performed to evaluate inflammatory changes in zebrafish brain tissues. These assessments were used to determine the conditions for establishing the zebrafish migraine model. After the model was established, zebrafish were assigned to Tianshu Capsules treatment groups. Changes in locomotor behavior and brain tissue biomarkers were evaluated to investigate the pharmacodynamic effects of Tianshu Capsules and further validate the model. The results showed that the locomotor velocity of zebrafish increased significantly after intraperitoneal injection of a 500 μg/mL nitroglycerin solution (P< 0.01). Brain tissue DA levels decreased significantly (P<0.01), the mRNA expression levels of il-1b, nos2b, c-fos, and c-jun were upregulated, and inflammatory cell infiltration increased, indicating that the zebrafish migraine model was successfully established. Treatment with Tianshu Capsules reduced the locomotor distance and velocity of zebrafish following nitroglycerin injection (P<0.001) and significantly decreased the mRNA expression level of il-1b in brain tissues (P<0.01). It also showed a tendency to reduce the mRNA expression levels of nos2b, c-fos, and c-jun. HE staining showed that inflammatory cell infiltration in zebrafish brain tissues was reduced after treatment with Tianshu Capsules compared with that in the model group. These findings demonstrate that a zebrafish migraine model can be successfully established by nitroglycerin injection and that Tianshu Capsules exert therapeutic effects in this model, thereby further supporting the validity of the model. This study provides a useful tool for the high-throughput screening of antimigraine drugs.