INTRODUCTION: Altered metabolic pathways are critical in the progression of traumatic brain injury (TBI). Identifying differentially abundant metabolites (DAMs) from specific cell types can offer valuable diagnostic and prognostic insights. OBJECTIVE: This study aimed to characterize the metabolomic profile of injured human brain microvascular endothelial cells (hBMEC) at 2-, 12-, 24-, and 48 h post-injury. METHODS: Using an in vitro TBI model, we analyzed metabolites in cell culture media through a combination of direct injection mass spectrometry and a custom reverse-phase LC-MS/MS assay. We evaluated 644 metabolites at each time point. RESULTS: Phosphatidylcholines were significantly upregulated across all time intervals. At 2- and 12 h post-injury, the most significantly upregulated metabolites included sphingomyelin (OH) C22:1, ethylmalonic acid, and methylhistidine, while guanosine and the combination of butyric acid + isobutyric acid were the most downregulated. At 24 and 48 h, deoxyadenosine and inosine, respectively, emerged as the most upregulated metabolites, with butyric acid + isobutyric acid and quinoline-4-carboxylic acid showing the greatest downregulation. CONCLUSION: Metabolomic profiling identified various DAMs after traumatic injury that are linked to human endothelial dysfunction. Future experiments should expand the number of metabolites measured to determine the underlying signaling pathways.