Abstract: Objective To establish a method by high-performance liquid chromatography (HPLC) for determining the concentration of magnetic mitomycin C-polybutylcyanoacrylate nanoparticles in mouse plasma. Methods Chromatography was performed on a LiChroCART C18 (250 mm×4 mm, 5 μm) column with the mobile phase consisting of acetonitrile-NaAC (15:85), the flow rate of 1.0 ml/min, and the detection wavelength of 365 nm. Sample extraction was carried out with ethylacetate. Results The linear range of mouse plasma mitomycin C concentration was 0.04-1.00 μg/ml, and the linear equation of Y=16 388X-17.17 (r=0.999 8) was derived. Conclusion This method is very easy to operate and suits the need of perclinical pharmacokinetic studies of mitomycin-magnetic nanoparticles and yields accurate and precise results.