Lawsone is a biomolecule that belongs to the naphthoquinone class found in henna leaves. This quinone is widely used in cosmetic products being considered the oldest coloring agent applied for dyeing hair. Moreover, biological activities have been attributed to lawsone such as antibacterial, antifungal, and anticancer. However, these activities are mostly limited due to its low aqueous solubility and chemical instability. In this context, this study aimed to develop nanoemulsion containing lawsone (NE-law) and assess its in vitro cytotoxicity against cervical carcinoma cells. Emulsion phase inversion method was used to prepare nanoemulsion formulations (NE1, NE2, NE3, NE4, and NE-law). Following the formulation development, NE1 formulation was chosen to incorporate lawsone, presenting average droplet size about 45 nm with narrow size distribution (PdI <0.1) and negative zeta potential ( -13 mV). In TEM analyses, droplets were visualized with irregular spherical shapes, high homogeneity, and without aggregation. The incorporation efficiency of lawsone into NE was higher than 90 %, and in vitro release profile of this naphthoquinone from formulation was gradual and regular throughout the experiment. Nanoemulsified lawsone improved in vitro cytotoxicity on cervical carcinoma cells compared to free lawsone. Therefore, we prepared a stable lawsone-loaded NE using a reproducible emulsification method.