A simple HPLC method was established for the determination of entrapment efficiency of a new 5-FPE liposome formulation. Chromatographic separation was performed on a Kromasil 100 A C18 column (350 mm×4.6 mm, 5 μm). The mobile phase was consisted of methanol-water (58:42, v/v). The flow rate of mobile phase was set at 0.8 mL/min. The UV detection wavelength was 271 nm, and the column temperature was 30 ℃. The linear range of 5-FPE was from 0.8-12.8 μg/mL, r = 0.9999. The RSD of intm-day and inter-day precision were less than 2.97%. The average recovery was from 96.8%-104.6% with RSD less than 2.24%. The method was simple, rapid, accurate, and sensitive. It is suitable for the determination of entrapment efficiency of the 5-FPE liposome formulation.