This research aimed to develop and optimize a nanoemulsion-based formulation containing ceramide IIIB using phase-inversion composition for transdermal delivery. The effects of ethanol, propylene glycol (PG), and glycerol in octyldodecanol and Tween 80 systems on the size of the nanoemulsion region in the phase diagrams were investigated using water titration. Subsequently, ceramide IIIB loading was kept constant (0.05 wt%), and the proposed formulation and conditions were optimized via preliminary screening and experimental design. Factors such as octyldodecanol/(Tween 80:glycerol) weight ratio, water content, temperature, addition rate, and mixing rate were investigated in the preliminary screening experiment. Response surface methodology was employed to study the effect of water content (30%–70%, w/w), mixing rate (400–720 rpm), temperature (20°C–60°C), and addition rate (0.3–1.8 mL/min) on droplet size and polydispersity index. The mathematical model showed that the optimum formulation and conditions for preparation of ceramide IIIB nanoemulsion with desirable criteria were a temperature of 41.49°C, addition rate of 1.74 mL/min, water content of 55.08 wt%, and mixing rate of 720 rpm. Under optimum formulation conditions, the corresponding predicted response values for droplet size and polydispersity index were 15.51 nm and 0.12, respectively, which showed excellent agreement with the actual values (15.8 nm and 0.108, respectively), with no significant (
P
>0.05) differences.