Teicoplanin is a glycopeptide antibiotic used for treatment of severe Gram-positive bacterial infection. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for therapeutic drug monitoring (TDM) of teicoplanin and to review our clinical experience.We established an LC–MS/MS method to analyze serum concentration of teicoplanin using simple protein precipitation with a 5 min run time for each sample. The linearity, lower limit of quantitation, detection accuracy, precision, carryover, matrix effect, and extraction recovery were evaluated. From September 2014 to June 2017, a total of 421 serum teicoplanin concentrations was measured in 223 patients. We collected demographic and clinical data, medication history, and laboratory findings through retrospective review of medical records.The LC–MS/MS method was linear for serum teicoplanin concentrations in the range of 12.0–89.0 g/mL. The intra- and inter-assay precisions were below CV 7.5%. The accuracy was less than ±10% bias. The lower limit of quantification was 0.2 g/mL. The extraction recovery ranged from 88.8% to 96.6%. Of 421 measurements, 87 (20.7%) were subtherapeutic (< 10 μg/mL), and four (0.9%) were above the toxic threshold (≥ 60 μg/mL). Serum teicoplanin concentration was measured once in 140 patients (63%), and multiple measurements were completed for the others (83 patients, 37%). Intra-patient variability in teicoplanin concentration was found (CV 33%, range 2–94%).Our simple and rapid LC–MS/MS method was successfully applied in TDM of teicoplanin in clinical practice. Such TDM of teicoplanin may be useful for individualized dose adjustment.