Abstract：Objective To evaluate percutaneous absorption in vitro and intradermal retention of original mupirocin ointment and generic mupirocin ointment. Methods Franz type diffusion cells were used for permeation studies in vitro. Excised and dorsal skins of KM mice were used as the transdermal barrier with the 0.1mol/L sodium dihydrogen phosphate solution (pH6.3) as the hydrophilic release solution and the mixed solution with 30% ethanol and 0.1mol/L sodium dihydrogen phosphate solution (pH6.3) as the lipophilic release solution. The concentrations of the release solution were determined by HPLC to investigate the cumulative permeation quantities at different times, and intradermal retention quantities were determined at the sampling end point. Results The percutaneous absorption percentage of mupirocin ointment produced by different enterprises in the hydrophilic release solution for 8h ranged from 0.01% to 0.05%, and the intradermal retention percentage ranged from 0.51% to 1.79%. The percentage range of 8h percutaneous absorption and intradermal retention in the lipophilic release solution was 0.01%~1.04% and 0.69%~2.59%, respectively. Conclusion The in vitro percutaneous release trend of the original preparation of mupirocin ointment was consistent with that of the generic preparation of mupirocin ointment. The percutaneous absorption and intradermal retention of the original medicine and the generic medicine in the lipophilic release solution were significantly different. This study can provide a reference for the consistency evaluation of the generic medicine of mupirocin ointment.