Abstract: OBJECTIVE To develop a LC-MS/MS method for determination of arotinolol concentration in human-plasma. METHODS The plasma samples were processed with ethyl acetate extraction and separated on ZORBAX Extend C₁₈ column (2.1 mm×100 mm, 3.5 μm) and eluted with acetonitrile-water (80∶20, containing 0.1% formic acid). Detection of the analyte was achieved using positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 372.3→m/z 315.9 and m/z 267.2→m/z 145.0 for arotinolol and internal standard, respectively. RESULTS The linear calibration curve of arotinolol was obtained concentration range of 0.5-1 000 ng·mL^-1(r=0.995 2), with the lower limit of quantitation of 0.5 ng·mL^-1. Intra-day and inter-day relative standard deviations were both <15%. The recoveries of low, middle and high concentrations were (80.6±1.6)%, (83.2±3.1)% and (87.5±4.5)%, respectively. CONCLUSION The established method is rapid, sensitive, accurate, specific and reliable, and suitable for the determination of arotinolol in human plasma and pharmacokinetic study.