Abstract: OBJECTIVE To establish a rapid and sensitive HPLC-MS/MS method for tosufloxacin in plasma. METHODS Tosufloxacin was extracted with acetonitrile and the residues were analyzed with a HPLC-MS/MS system used Agilent Eclipse Plus C₁₈ column(4.6 mm×150 mm, 3.5 μm) with the mobile phase consisted of acetonitrile (0.1% formic acid) with 5 mmol·L^-1 ammonium formate(36∶64), with a flow rate of 0.5 mL·min^-1 and temperature of 40 ℃. Multiple reaction monitoring(MRM) using the precursor to production combinations of m/z 405.2→387.2 and m/z 332.2→314.2 was performed to detect tosufloxacin and the internal standard, respectively. RESULTS The calibration curves for tosufloxacin had good linearity within the range of 6-2 000 ng·mL^-1, r=0.996 2. The limits of quantitation for tosufloxacin was 6 ng·mL^-1. The intra-day and inter-day precision of variation was <10%. The average recoveries for high-, middle-, low-dose of tosufloxacin(1 800, 750, 15 ng·mL^-1) were 97.5%, 103.3% and 105.9%, respectively. CONCLUSION The method provides a sensitive, accurate, precise and reliable analytical procedure for clinical monitoring of tosufloxacin plasma and its pharmacokinetic studies.