This paper describes a novel technique to determine mass diffusion coefficients of multi-component system within a transparent mixture by using an optical system. The mixture is composed of sodium chloride and lysozyme as solutes. Binary and multi-component solution experiments were conducted separately under constant temperature conditions. By measuring transient diffusion fields inside the cell, as well as for mixed multi-component solutions, it was confirmed that within the concentration ranges considered in this study, the diffusion of each solute inside the cell progresses independently. This indicates the superposition principle of concentration for certain levels of sodium chloride and lysozyme within the cell. Furthermore, by using this concentration superposition principle and an inverse analysis based on the conjugate gradient method, the diffusion coefficients of each solute in the mixture were successfully obtained from several multi-component experiments. Each obtained diffusion coefficient was good agreement with the determined diffusion coefficient from binary experiment.