In this review, the evaluation methods for the screening of depigmenting substrates were investigated. For this purpose, the evaluation method of tyrosinase, a key enzyme of melanin biosynthesis, is most frequently used, but evaluating methods based on the regulation of cellular signal transfer factors or the inhibition of melanosome transfer have also been developed. Evaluation of the depigmenting effect using melanocytes is complex. It has the advantage of being capable of analysing overall effects on melanin biosynthesis at cellular levels. Before the final clinical testing of depigmenting agents, in vitro testing should be conducted to confirm the depigmenting efficacy and safety. Clinical studies for depigmenting agents can be used to investigate the prevention of melanin biosynthesis and to determine whether melanin disappears from skin. Therefore, the most appropriate protocol has to be employed, depending on the mechanism of action of the depigmenting agent.