Background and Objectives: Tissue optical clearing technique based on immersion of tissues into optical clearing agents (OCAs) can reduce the scattering and enhance the penetration of light in tissue. However, the barrier function of epidermis limits the penetration of OCAs, and hence is responsible for the poor optical clearing efficacy of skin by topical action. In this study, a variety of light irradiation was applied to increase permeability of agents in skin and improve the optical clearing efficacy. Study Design/Materials and Methods: Different light sources with different dose, i.e, CO 2 laser, Nd:YAG laser (532 and 1,064 nm) with different pulse modes and Intense Pulsed Light (IPL) (400-700 and 560-950 nm) were used to irradiate rat skin in vivo, and then glycerol was applied onto the irradiated zone. VIS-NIR spectrometer was utilized to monitor the changes of reflectance. In vitro skin samples were also irradiated by Q-switched Nd:YAG laser (1,064 nm) and then treated by glycerol for 10-60 minutes. Based on the measurement of the reflectance and transmittance of the samples, the optical properties of skin and penetration depth of light were calculated. Results: Results show that photo-irradiation with appropriate dose combining with the following glycerol treatment is able to reduce in vivo skin reflectance. Compared with the control group, the maximal changes in reflectance are ninefold at 575 nm and eightfold at 615 nm, respectively, which were caused by Q-switched 1,064-nm Nd:YAG laser irradiation and following glycerol treatment. The results for in vitro skin demonstrate that the joint action can significantly increase the optical penetration depth in samples. Conclusions: The combination of Q-switched Nd:YAG (1,064 nm) laser and glycerol could enhance optical skin clearing efficacy significantly. This study provides a noninvasive way to improve the optical clearing of skin, which will benefit the skin optical therapy.