Abstract Background This study aimed to establish a method to determine tigecycline (TGC) in cerebrospinal fluid (CSF) and serum of 12 patients with Multidrug-resistant acinetobacter baumannii (MDRAB) central nervous system infection (CNSI) and evaluate the correlation of TGC in CSF and serum samples. Methods TGC in CSF and serum was extracted by acetonitrile and detected by HPLC-MS/MS. The separation was performed on the Waters XBridge® BEH Shield RP18 column. This method could achieve the quantification accurately in a very short time. Results For these 12 patients, the trough concentration ranges of TGC in CSF and serum at steady-state were 16.35-53.56 ng/mL and 67.76-211.9 ng/mL. The CSF-to-serum ratio of TGC at steady-state trough concentration was ranged from 21.46% to 44.46%, and the mean value was 31.61 ± 8.13%. The correlation of TGC in CSF and serum was 0.5065. CNSI might have no potential to increase the penetration ability of TGC to CSF. Conclusion The method was validated to be accurate and effective. The correlation between the concentrations of TGC in CSF and serum at steady-state was demonstrated to be positive based on quantification of TGC in bio-samples from 12 MDRAB patients.