Solid microneedles (MNs) represent a useful tool for enhancing skin permeability by creating microchannels that provide a drug delivery route. To achieve the solid polymer MNs to become a clinical reality and to be commercialised, it is much essential to understand the skin penetration process. In this work, the effect of polymer MN height and density, drug molecular weight, as well as drug diffusion time on the drug permeability distribution was systemically investigated in vivo. MN with a height of 800 mu m was most conductive to enhance the vertical distribution of drug permeation into the skin, while 11 x 11 MN array was most beneficial to promote the horizontal distribution of drug permeation into the skin. In addition, the increasing of drug molecular weight could reduce the drug permeability distribution and Fluorescein isothiocyanate most likely to penetrate into the skin after MNs pre-treatment. With the increase of drug diffusion time, the drug distribution in the subcutaneous gradually weakened until the drug was absorbed by the subcutaneous tissue at 8 h. These results suggest that the solid polymer MNs can penetrate the stratum corneum of the skin for enhancing drug delivery, especially small molecule drugs.