Rheumatoid arthritis (RA) is a debilitating autoimmune disease that causes progressive chronic inflamma tion of the joints and destruction of articular cartilage and bone erosion. Cartilage destruction is a key characteristic in patients with RA. RA fibroblast-like synoviocytes (FLS) mainly contributes to local production of cytokines, inflam matory mediators and MMPs, and to migrate and destruct joint cartilage. Here, we summarized a detailed protocol for developing a humanized synovitis animal model. A cartilage-sponge complex without RA FLS was implanted under the left flank skin of a SCID mouse primarily, two weeks later, cartilage-sponge complex containing RA FLS was inserted under the right skin of the contralateral flank. The H&E staining clearly helps to identify the cartilage damage on the day 45 after second implantation. This model is highly significant to investigate the role and mecha nisms of agents or cells in targeting RA FLS in vivo.