Tyrosinase is a key enzyme that catalyzes melanogenesis in human skin. It oxidizes tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) and subsequently to dopachrome, which further polymerizes to melanin pigments. Therefore finding an effective tyrosinase inhibitor, either from synthetic or natural sources, is not only useful as skin whitening agents in cosmetic application, but also beneficial in treating melanin-related disorders. The present study reports of the optimized and validated results of a cell-based tyrosinase assay using B16F10 murine melanoma cell line, which produces melanin pigments and has been used extensively in antimelanogenesis studies. The optimization studies involved 3 parameters (1) optimal seeding cell number per well for total protein extraction; (2) optimal dopachrome formation from enzymatic reaction between total protein (tyrosinase source) and L-DOPA (substrate); and (3) optimal incubation period after the addition of substrate. The present study demonstrates that using seeding cell number of 2 × 105 cells/well, total protein of 40 μg, L-Dopa of 5 mM,and at an incubation period of 1 hour at 37°C provided the optimal response on cultured melanoma cells. Kojic acid, a standard tyrosinase inhibitor, was used as a positive control in the optimized cell-based tyrosinase assay to validate the usefulness of the assay. CONCLUSION: The use of the mentioned protocol is sensitive to determine changes in melanoma cells as the result of tyrosine inhibitors.