Red blood cells or erythrocytes, constituting 40 to 45 percent of the total volume of human blood are vesicles filled with hemoglobin with a fluid-like lipid bilayer membrane connected to a 2D spectrin network. The shape, volume, hemoglobin mass, and membrane stiffness of RBCs are important characteristics that influence their ability to circulate through the body and transport oxygen to tissues. In this study, we show that a simple two-LED set up in conjunction with standard microscope imaging can accurately determine the physical and mechanical properties of single RBCs. The Beer–Lambert law and undulatory motion dynamics of the membrane have been used to measure the total volume, hemoglobin mass, membrane tension coefficient, and bending modulus of RBCs.