Cerebral malaria (CM) is a severe neurological condition caused by Plasmodium falciparum. Disruption of the brain-blood barrier (BBB) is a key pathological event leading to brain edema and vascular leakage in both humans and in the mouse model of CM. Interactions of brain endothelial cells with infected red blood cells (iRBCs) and with circulating inflammatory mediators and immune cells contribute to BBB dysfunction in CM. Adjunctive therapies for CM aim at preserving the BBB to prevent neurologic deficits. Experimental animal and cellular models are essential to develop new therapeutic strategies. However, in mice, the disease develops rapidly, which offers a very narrow time window for testing the therapeutic potential of drugs acting in the BBB. Here, we establish a brain endothelial cell barrier whose disturbance can be monitored by several parameters. Using this system, we found that incubation with iRBCs and with extracellular particles (EPs) released by iRBCs changes endothelial cell morphology, decreases the tight junction protein zonula occludens-1 (ZO-1), increases the gene expression of the intercellular adhesion molecule 1 (ICAM-1), and induces a significant reduction in transendothelial electrical resistance (TEER) with increased permeability. We propose this in vitro experimental setup as a straightforward tool to investigate molecular interactions and pathways causing endothelial barrier dysfunction and to test compounds that may target BBB and be effective against CM. A pre-selection of the effective compounds that strengthen the resistance of the brain endothelial cell barrier to Plasmodium-induced blood factors in vitro may increase the likelihood of their efficacy in preclinical disease mouse models of CM and in subsequent clinical trials with patients.