Calcium ions (Ca2+) are crucial as universal second messenger signaling molecules in numerous cellular processes. In the cardiovascular system, intracellular calcium signaling is important for regulating vascular resistance, functional hyperemia, leukocyte transmigration, and coagulation. In vitro and in vivo live calcium imaging is a pivotal technique in modern cellular biology, allowing the visualization of intracellular calcium dynamics with high temporal and spatial resolution. Calcium imaging is particularly significant for studying the downstream effects of ion channels, G protein-coupled receptors (GPCRs), and other signaling pathways that modulate cellular calcium levels. Here, we describe a simple and fast method of investigating the intracellular calcium changes in an in vitro blood–brain barrier model by utilizing the fluorescent dye Fluo-4 AM in primary brain endothelial cells.