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Cardiovascular diseases are the leading cause of death worldwide and are not typically diagnosed until the disease has manifested. Endothelial dysfunction is an early, reversible precursor in the irreversible development of cardiovascular diseases and is characterized by a decrease in nitric oxide production. We believe that more reliable and reproducible methods are necessary for the detection of endothelial dysfunction. Both nitric oxide and calcium play important roles in the endothelial function. Here we review different types of molecular sensors used in biological settings. Next, we review the current nitric oxide and calcium sensors available. Finally, we review methods for using both sensors for the detection of endothelial dysfunction.