The blood-brain barrier (BBB) separates the peripheral circulation from the brain. Brain endothelial cells (BECs) line the inner lumen of blood vessels in the brain and express tight junction proteins, that prevent the leakage of molecules between the cells into the brain, and efflux transporters that actively pump molecules out of the brain. As we age, the BBB becomes leaky and dysfunctional, leading to cerebrovascular diseases and dementia. We here investigated how BECs become senescent, a major feature of aging, and change their metabolic profile. First, we demonstrate that both the chemotherapeutic doxorubicin, a known inducer of cellular senescence, as well as hydrogen peroxide, a mediator of oxidative stress, induce BEC senescence. This was indicated by positive senescence-associated β-galactosidase staining (P = 0.002 and P = 0.001 for doxorubicin and H2O2, respectively) and increased P16 gene expression (both P = 0.004). Next, we used mass spectrometry and identified 74 differentially changed metabolites (P < 0.05) in doxorubicin-induced and 66 in H2O2-induced senescent cells. 33 were present in both treatments (Jaccard index of 0.236). Of those, 13 were already reported in the literature to be changed during senescence in the brain or other organs. We also identify new metabolites and provide pathway analyses of the differentially changed metabolites. These analyses are the first to characterize BBB cell senescence at a metabolite level and provide the basis for the development of novel therapeutic interventions for age-related neurological disorders.