Nanoemulsions (NE) are lipid nanocarriers that can efficiently load hydrophobic active compounds, like palmitoyl-L-carnitine (pC), used here as model molecule. The use of design of experiments (DoE) approach is a useful tool to develop NE with optimized properties, requiring less experiments compared to trial-and-error approach. The optimized formulation was studied for its stability, scalability, pC entrapment, loading capacity, and biodistribution in mice. NE were prepared by the solvent injection technique. DoE was implemented for designing pC-loaded NE, using a two-level fractional factorial design as model. NE were fully characterized by a combination of techniques, and biodistribution was studied ex-vivo after injection of fluorescent NE in healthy mice. We selected the optimal formulation of NE, named pC-NEU, after DoE analysis of four variables. pC-NEU incorporated pC in a very efficient manner, with high entrapment efficiency and loading capacity. pC-NEU did not change its initial colloidal properties stored at 4ºC in water during 120 days, nor in buffers with different pH values (5.3 and 7.4) during 30 days. The scalability process did not affect NE stability properties. Biodistribution study showed that pC-NEU formulation was predominantly concentrated in the liver, with minimal accumulation in spleen, stomach and kidneys.