OBJECTIVE：To develop a metho d for determining the plasma concentration of sulfasalazine （SSZ）metabolite sulfapyridine（SP）in rats ，and to investigate the effects of esomeprazole （ESOM）on the pharmacokinetic behavior of SSZ in rats. METHODS：Male SD rats were randomly divided into SSZ group and SSZ+ESOM group ，with 6 rats in each group. SSZ+ESOM group were given Esomeprazole enteric-coated tablets [ 90 mg/（kg·d）] intragastrically for 14 days. On the 15th day ，the rats in 2 groups were given Sulfasalazine enteric coated tablets （90 mg/kg）intragastrically，and blood sample was collected from the inner canthus at 0.5，1，1.5，2，3，4，6，8，10，12，24，36，48，72 h after administration. After protein precipitation with methanol ， using diazepam as internal standard ，Agilent XDR-C 18 column was adopted with methanol- 0.1％ formic acid solution （gradient elution）as mobile phase. The concentration of SSZ metabolite SP in plasma was determined by LC-MS/MS. The pharmacokinetic parameters were calculated by using DAS 3.0.1 software and compared between 2 groups. RESULTS ：The linear range of SP were 2-1 000 ng/mL. The methodology met the requirements of Chinese Pharmacopeia . There was no statistical significance in pharmacokinetic parameters of SP between 2 groups，such as AUC 0－t，tmax，t1/2z，cmax，MRT0－t（P＞0.05）. CONCLUSIONS ：The established method is simple ，rapid and sensitive ；it can be used for the concentration determination of SSZ metabolite SP in plasma. ESOM has no significant effect on the pharmacokinetic behavior of SSZ in rats.