Abstract: OBJECTIVE To develop a rapid, highly sensitive and selective performance LC-MS/MS for the determination of vancomycin in rabbit cerebrospinal fluid. Explore and complete the pharmacokinetic study of vancomycin in rabbits. METHODS The concentration of vancomycin in the cerebrospinal fluid was observed after intravenous injection of vancomycin in rabbits and intragastric administration of borneol followed by intravenous injection of vancomycin. Internal standard method was used in analysis to quantify the vancomycincerebrospinal fluid concentration. DAS software was used to obtain kinetic parameters such as AUC, C_max, and T_1/2. RESULTS The inter-day precision of vancomycin in rabbit cerebrospinal fluid was in the range of 4.7%-6.1%, the intra-day precision was in the range of 1.4%-7.1% and the accuracy was in the range of 93.6%-106.0%, respectively. Linear calibration curves for vancomycin was observed at 50-5 000 ng·mL^-1 with correlation coefficient r >0.99. The AUC and C_max values of vancomycin in borneol and vancomycin administrated group were both significantly higher than that in vancomycin single administrated group. CONCLUSION This method is successfully applied in the rabbit cerebrospinal fluid concentration determination of vancomycin. Borneol can improve the ability of vancomycin to penetrate the blood brain barrier.