Abstract: The aim of the study was to develop a simple, rapid and accurate LC-MS/MS method for the determination of digoxin.Digoxin-d3 was taken as the internal standard (IS), and sample preparation was achieved by liquid-liquid extraction.Chromatographic separation was performed on a Kinetex C₁₈ column (2.1 mm × 50 mm, 2.6 μm; Phenomenex) using an isocratic elution with merely 2 min for each sample.The mobile phase consisted of water and acetonitrile solutions, both containing 1 mmol/L ammonium acetate and 1 mmol/L formic acid (55∶45).The detection was conducted on a TripleQuad^TM 4500MD mass spectrometer coupled with electrospray ionization interface under positive-ion multiple reaction monitoring mode.The transitions were m/z 798.5 → 651.3 and m/z 801.6 → 654.4 for digoxin and digoxin-d3, respectively.Results showed that the method was linear over the range of 0.100-20.0 ng/mL.The selectivity, accuracy and precision, recovery and stability of the method were all within the acceptable limits with no matrix effect.This method was successfully applied to a girl treated with digoxin with substantial improvement of therapeutic effect and elimination of toxic reaction, so it can provide valuable fuidance and reference for individualized medication in clinical practice.