Abstract: OBJECTIVE To investigate the material basis of Dunhuang Xiaobi Zhitong tincture in the treatment of osteoarthritis(OA) using a “dry and wet” strategy, consisting of computer aided drug design such as transdermal permeability calculation, complex network, molecular docking, molecular dynamics simulation, combined with chemical analysis, enzyme inhibition assay and experiments in vitro. METHODS Select transdermal compounds from traditional Chinese medicine that had been verified by experiments. The skin permeability parameters were calculated by using Qikprop module of Schrödinger software. The range of transdermal parameters was established and the qualified components in Dunhuang Xiaobi Zhitong tincture were screened. Efficacy groups were divided according to Dunhuang Xiaobi Zhitong Tincture treatment efficacy, and potential targets and KEGG pathways for OA treatment in different efficacy groups were explored through reverse prediction of targets, complex network analysis and KEGG pathway enrichment analysis. The molecular docking method was used to virtually screen the components in the target COX-2 partner that met the range of skin permeability parameters, and the compounds were selected for enzyme activity assay, ADMET calculation, skin permeability assay, cell experiments, and explant experiments. RESULTS The 69 potential transdermal compounds were obtained. Complex network analysis showed that the targets of OA treatment in the promoting blood circulation group were mainly VEGFA, IGF1R and HIF-1α. The main KEGG signaling pathways were Ras and RAP1 signaling pathways, etc. The targets of OA treatment in the resolving heat group were mainly TLR9, IL-6 and ADAMTS5. The main KEGG signaling pathways were TNF, NF-κB signaling pathway, etc. The results of macromolecular docking-enzyme activity-cell experiment showed that, cimifugin, imperatorin, and vanillic acid could inhibit COX-2 activity and inhibit the synthesis of PGE2 in chondrocytes. Cimifugin could effectively relieve the damage of cartilage damage in cartilage explant models. The transdermal absorption experiment showed that the cumulative permeability per unit area of cimifugin in 12 h was 128.1 μg·cm. The average transdermal accumulation rates was 28.2%. The result of molecular dynamics simulation showed that cimifugin formed a relatively stable hydrophobic interaction with PHE 518 and formed stable hydrogen bond with the residues TYR 355, LEU 384 and SER 530. CONCLUSION In this study, a “dry and wet” combination strategy of computer aided drug design combines with a variety of chemical and biological experiment are used to elucidate the material basis of Dunhuang Xiaobi Zhitong tincture in the treatment of OA. This work may provide methodological reference for the study of external preparation of traditional Chinese medicine.