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Cell extraction_LC–MS strategy was applied to screen and identify potential neuroprotective compounds from S. buergeriana by using HT22 cells.Two neuroprotective metabolites, buergeriside C1 and buergeriside A1, were selectively bound HT22 cells.The significant metabolites, which are compounds that affect the statistical difference between the two groups, were identified.The cell extraction approach is a better representation of the in vivo situation than other methods.In the cell extraction_LC–MS method, when cells are incubated with natural product extracts, bioactive compounds selectively bind to extracellular or intracellular targets. The extracts and major compounds (phenylpropanoids and iridoid glycosides) of Scrophularia buergeriana Miquel have been reported to show neuroprotective effects both in vitro and in vivo. In this study, the cell extraction_LC–MS strategy was applied to screen and identify potential neuroprotective compounds from S. buergeriana by using immortalized mouse hippocampal HT22 cells. The results showed that two known compounds from S. buergeriana selectively bound HT22 cells. Additionally, metabolomics analyses were performed using the Mass Profiler Professional and Limma differential expression package of R to identify significant differences between HT22 cells treated with S. buergeriana and untreated cells. The cell extraction approach more accurately reflects in vivo conditions compared with other methods and can be readily used for screening bioactive components from natural products.