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Sharifah Nurfadhlin Afifah Syed Azhar, Siti Efliza Ashari, Norazlinaliza Salim Integrated Chemical BioPhysics Research, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia Introduction: Kojic monooleate (KMO) is an ester derived from a fungal metabolite of kojic acid with monounsaturated fatty acid, oleic acid, which contains tyrosinase inhibitor to treat skin disorders such as hyperpigmentation. In this study, KMO was formulated in an oil-in-water nanoemulsion as a carrier for better penetration into the skin.Methods: The nanoemulsion was prepared by using high and low energy emulsification technique. D-optimal mixture experimental design was generated as a tool for optimizing the composition of nanoemulsions suitable for topical delivery systems. Effects of formulation variables including KMO (2.0%–10.0% w/w), mixture of castor oil (CO):lemon essential oil (LO; 9:1) (1.0%–5.0% w/w), Tween 80 (1.0%–4.0% w/w), xanthan gum (0.5%–1.5% w/w), and deionized water (78.8%–94.8% w/w), on droplet size as a response were determined.Results: Analysis of variance showed that the fitness of the quadratic polynomial fits the experimental data with F-value (2,479.87), a low P-value (P100 µg/mL.Conclusion: The survival rate of 3T3 cell on KMO formulation (54.76%) was found to be higher compared to KMO oil (53.37%) without any toxicity sign. This proved that the KMO formulation was less toxic and can be applied for cosmeceutical applications. Keywords: kojic monooleate, anti-tyrosinase, hyperpigmentation, nano-cosmeceutical, D-optimal mixture experimental design