Blood-brain barrier (BBB) dysfunction is a well-established pathological phenotype of ischemic stroke, and targeting BBB integrity has emerged as a promising therapeutic strategy. Danshen-Chuanxiong formula (DS-CX), an effective herbal combination against ischemic stroke, has demonstrated regulatory effects on the BBB at various stages of ischemic stroke. However, its specific BBB-protective components and underlying molecular mechanisms remain unclear. Recent advances in multicellular self-assembled BBB spheroids have shown distinct advantages in disease modeling and drug discovery, offering a novel approach to address these questions. To simulate ischemic stroke-induced BBB dysfunction, we developed an oxygen-glucose deprivation/reoxygenation (OGD/R)-induced BBB disruption model using multicellular spheroids. To identify the effective substances of DS-CX responsible for BBB protection, we conducted a multi-parametric evaluation to assess BBB permeability, tight junctions, cell viability, reactive oxygen species (ROS) levels, inflammatory markers, and apoptotic phenotypes using high-content imaging. Further immunofluorescence and transcription analyses were performed to elucidate the BBB-protective mechanisms of DS-CX and its active components. Similar to the overall effects of DS-CX on BBB protection, preliminary screening fortunately found that both protocatechuic acid, ferulic acid, and senkyunolide I significantly reduced OGD/R-induced leakage, and upregulated the protein and mRNA levels of ZO-1 and Claudin-5 in BBB spheroids. Further multi-phenotypic assessments manifested that DS-CX and its active compounds effectively improved cell survival, reduced ROS production, inhibited inflammation, and decreased apoptosis, compared to the damaged BBB spheroids without drug intervention. Molecular experiments showed that DS-CX and its active constituents not only rescued the abnormal protein levels of pivotal targets related to oxidative stress (HO-1), inflammation (MMP-9, TLR-4), and apoptosis (Caspase-3, Bax, Bcl-2) in OGD/R-treated BBB spheroids, but also normalized the dysregulated mRNA levels of vWF, HO-1, MMP-9, TLR-4, TNF-α, IL-6, IL-1β, and IL-18 caused by OGD/R stimulation. Collectively, the present work successfully identified protocatechuic acid, ferulic acid, and senkyunolide I as key BBB-protective components of DS-CX against ischemic stroke. These compounds likely exert their therapeutic effects through multi-target regulation of oxidative stress, inflammation, and apoptosis. Our findings provide a novel spheroid-based multi-parametric screening approach for discovering BBB-targeted therapies in ischemic stroke.