The present investigation was designed with an aim to formulate Chitosan (CH) films by cross-linking with Sodium citrate (Nacit), Sodium tripolyphosphate (NaTPP) or Chondroitin sulphate (CS) that shall be capable of simulating the in vitro permeation of model polar drug, 5-Fluorouracil (5-FU) and non polar drug, Indomethacin (INDO) across rat/rabbit or human epidermis. Optimization design was employed to identify and optimize various process and formulation variables significantly (p<0.05) influencing permeation of both drugs across these films. Further, the results were evaluated for the effect of cross linking density on in vitro permeation using atomic absorption spectroscopic analysis for Na+, DSC and IR spectroscopic analysis. The in vitro permeation of both 5-FU and INDO across optimized film formulations of films crosslinked with CH-NaTPP, CH-Nacit or CH-CS was found to be comparable to that obtained across rat, rabbit and human epidermal sheets. These results indicate that these optimized CH films have a potential to be developed as substitute for animal and human cadaver epidermal sheets for preliminary in vitro permeation studies