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Quercetin (QCT) is a natural polyphenolic flavonoid showing great potential in the treatment of skin cancer. However, its use is limited owing to its poor water solubility, poor absorption, quick metabolism and excretion, as well as low stability. Preparation of nanoemulgel has been proven to be an effective approach to deliver the drugs topically due to various advantages associated with it. This study aimed to prepare stable nanoemulgel of QCT using a Design-of-Experiments (DoE) tool for optimization, to characterize and to assess its in vivo toxicity and efficacy against human cancer cell lines in vitro. An ultrasonication emulsification method was used for the preparation of QCT-loaded nanoemulsion (QCT@NE). Box–Behnken design was used for the optimization of developed nanoemulgel. Then, in vitro characterization of prepared nanoemulsion was performed using Fourier Transform-Infra Red (FT-IR) spectroscopy, Scanning Electron Microscopy (SEM), particle size analysis, determination of zeta potential and entrapment efficiency (%EE). Further, the developed QCT-loaded nanoemulgel (QCT@NG) was characterized in vitro using texture profile analysis, viscosity and pH determination. Eventually, the cell cytotoxicity studies of the prepared nanoemulgel were performed on the skin cancer cell lines A431 followed by an acute toxicity and skin irritation study on male wistar rats in vivo. The developed QCT@NE was found to be nanometric in size (173.1 ± 1.2 nm) with low polydispersity index (0.353 ± 0.13), zeta potential (−36.1 ± 5.9 mV), and showed good %EE (90.26%). The QCT@NG was found to …