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The purpose of this study was to investigate whitening agent from a natural substance using in vivo model zebrafish. Tyrosinase is a key enzyme in melanogensis which was used for screening of whitening agent with tyrosinase inhibition assay and melanocyte cell melanin biosynthesis inhibition assay. However, pigmentation mechanisms were very a complicated procedure in human dermis. In this study, we used zebrafish embryo as an animal model for confirmed phenotype-based screening of melanin synthesis with natural sources. Zebrafish has most of same organs and tissues that can be seen in human. Especially, pigments were located in the epidermal layer on the surface of organism. In order to examine the whitening effects involved in pigment cell signaling, we treated zebrafish embryos with 10 kinds of methanol extracts of medicinal herbs. Among those medicinal herbs tested, 50 μg/mL of MeOH extract of Forsythia suspensa was decreased the pigmentation on zebrafish larva. After solvent partitioning with MeOH extract of F. suspense , chloroform and ethyl acetate fractions showed the depigmentation effect on zebrafish. In contrast the tyrosinase inhibition effect of F. suspense has not shown. Those results suggest that different approaches to obtain depigmentation process, and confirm the new protocols to evaluate mleanogenesis using in vivo zebrafish model.