In order to enhance the optical clearing effect of topically applied optical clearing agents (OCAs), we evaluated the effect of propylene glycol (PG) as a chemical penetration enhancer (PE) on optical clearing of skin in vitro by observation and measurement of optical-transmittance and diffuse-reflectance spectra. Three OCAs, i.e., glycerol, D-sorbitol and PEG400, and two other penetration enhancers, Azone and Thiazone, were used in this study. The results indicated that the decrease of reduced scattering coefficient caused by OCA/PG was larger than that by pure OCA, and the change by OCA/water was the least after the same treatment time. There were significant differences for the reduced scattering coefficient at 630 nm after 120 min application of agents between OCA and OCA/PG. The efficacy of optical clearing caused by OCA/PG depended on the OCA itself. When PEG400 was mixed with three different PEs, we found the optical clearing were different. The penetration enhancing ability of PG was much better compared to Azone, and suboptimal to Thiazone. Also, this study provides evidence for the use of PG as a PE in order to improve skin optical clearing.