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Mycoplasma synoviae(MS) infection is a serious threat to poultry industry in China, thus it is essential to study the pharmacokinetics (PK) in the target site of MS-infected chickens, but there are no relevant reports at present. The aim of this study was to compare the PK of tilmicosin in plasma and joint dialysate in MS-infected chickens. The MS infection model was established by evaluating the influence factors of the susceptibility of chicken species, day age of chicken, infection routes, infection cycle, infection dose, and stress response. The clinical symptoms, pathogen isolation, PCR identification, and ELISA antibody were detected to determine whether the MS infection model has been successfully established. Eight-week-old Mahuang chickens were challenged with MS by joint combined with footpad, 2 mL each time, twice a day for 5 d, then the MS infection model was successfully established. The infection group was orally administrated a single dose of 15 mg/kgbody weight (b. w.) tilmicosin. The joint dialysate was collected by the microdialysis technique, then the concentration of tilmicosin in plasma samples and joint dialysate were determined by triple quadrupole high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). There was no significant difference in elimination half-life (t1/2) and the mean residence time (MRT) of dialysate and plasma. In contrast, the time of the area under the concentration-time curve (AUC) and the (maximum concentration of tilmicosin in plasma) Cmaxof tilmicosin in plasma was 2.1 and 1.4 times higher than in dialysate. The distribution coefficient of tilmicosin in joint and plasma (AUCdialysate/AUCplasma) was 0.51. In conclusion, tilmicosin concentration in joints of MS-infected chicken was much lower than that of plasma, which may result in the poor clinical effect and drug resistance. The study could provide a reference for the clinical use of tilmicosin against MS.