Display omitted •Stearic acid SLN were optimized by a 22 full factorial design for skin administration.•Stearic acid SLN were of ca. 200 nm mean size, with 0.200 of PdI and ZP values of |26 mV|.•Stearic acid SLN were non-toxic to NIH3T3 fibroblasts. The present study focuses on the characterization of the cytotoxic profile on NIH3T3 mouse embryonic fibroblasts of solid lipid nanoparticles (SLN) optimized by a 22 full factorial design for skin administration. To build up the surface response charts, a design of experiments (DoE) based on 2 independent variables was used to obtain an optimized formulation. The effect of the composition of lipid and water phases on the mean particle size (z-AVE), polydispersity index (PdI) and zeta potential (ZP) was studied. The developed formulations were composed of 5.0% of lipid phase (stearic acid (SA), behenic alcohol (BA) or a blend of SA:BA (1:1)) and 4.7% of surfactants (soybean phosphatidylcholine and poloxamer 407). In vitro cytotoxicity using NIH3T3 fibroblasts was performed by MTT reduction assay. This factorial design study has proven to be a useful tool in optimizing SLN (z-AVE ∼ 200 nm), which were shown to be non-cytotoxic. The present results highlight the benefit of applying statistical designs in the preparation and optimization of SLN formulations.