AbstractFlurbiprofen axetil (FA) is a prodrug of flurbiprofen (FP), and it is hydrolyzed to the active FP by carboxylesterase in plasma after intravenous injection. The pharmacological action of FP is closely related to its chirality, and S‐FP shows better analgesic effects than R‐FP. Therefore, it is of great significance to compare the in vivo pharmacokinetic behaviors of R‐FP and S‐FP. In this study, we designed a sensitive high performance liquid chromatography–tandem mass spectrometry method and used CHIRALPAK‐IG3 column for chiral separation to quantify the concentrations of R‐FP and S‐FP in rat plasma. The results show that this method can accurately and effectively analyze the contents of R‐FP and S‐FP in plasma. In addition, the systemic exposure was approximately 3.09‐folds for the S‐FP compared with the R‐FP following intravenous administration of the FA to rats at a single dose of 4.5 mg/kg. More importantly, the clearance rate of S‐FP is significantly smaller than that of R‐FP. Therefore, the development of S‐FA injectable emulsion for clinical treatment of postoperative pain is very necessary.