Previous studies have demonstrated the ultrasound-induced skin optical clearing enhancement with topical application of 60% glycerol (G) on in vitro porcine skin and in vivo human skin. Our purpose was to find the relation between the effect of optical skin clearing and different concentrations of glycerol and to find more effective ultrasound-glycerol combinations on optical skin clearing. The enhancement effect of ultrasound [Sonophoresis (SP) delivery] in combination with 40% G, 60% G, and 80% G on in vitro human skin optical clearing was investigated. Light imaging depths of skin were measured using optical coherence tomography. Different concentrations of glycerol and ultrasound with a frequency of 1 MHz and an intensity of 0.5 W/cm2 was simultaneously applied for 15 min. The results show that with the increase of concentration of glycerol, the optical clearing of skin is much improved. Optical clearing capability of glycerol was more enhanced with simultaneous application of ultrasound compared with glycerol alone. The attenuation coefficients of skin tissues after application of 40% G/SP, 60% G/SP, and 80% G/SP decreased approximately 11.8%, 18.5%, and 20.0% at 15 min compared with 40% G, 60% G, and 80% G alone, respectively. The greatest decrease in attenuation coefficients at 60 min was approximately 52.3% and 63.4% for 80% G (without ultrasound) and 80% G/SP (with ultrasound), respectively, which are 2.1-fold and 2.6-fold to that in the 40% G.