To explore the optimum preparation process of Kunbixiao Prescription in treating rheumatoid arthritis(RA).Methods:Kunbixiao Prescription was prepared with three different processes.The male and female rat models of collagen-induced arthritis(CIA) were induced by injection of bovine type Ⅱ collagen with complete Freund's adjuvant.The paw swelling volume，arthritis index，spleen index，and pathological changes in the knee joint were estimated as the indicators of the therapeutic effects on both male and female rats in different groups.The male and female mouse models of writhing-induced pain was established with acetic acid，and the pain-relieving effects of Kunbixiao Prescription prepared with different processes on both male and female mice were evaluated.Results:The Kunbixiao Prescription prepared with process 2 reduced the paw swelling volume and arthritis index in both male and female CIA rats(P<0.01; P<0.05).The Kunbixiao Prescription prepared with processes 2 and 3 reduced the spleen index of female CIA rats(P<0.01; P<0.05).The Kunbixiao Prescription prepared with process 2 mitigated synovial hyperplasia and inflammatory infiltration in male and female CIA rats(P<0.05; P<0.01) and reduced the times of writhing in male mice(P<0.01).The Kunbixiao Prescription prepared with process 3 reduced the times of writhing in both male and female mice(P<0.05).Conclusion:The Kunbixiao Prescription prepared with process 2 ameliorated the joint inflammation and pathological changes and relieved the pain in CIA rats.This study provides a basis for the following research on the preparation process of Kunbixiao Prescription.