Novel anti-resistant liposomes have been developed to overcome intrinsic resistance in leukemia.Anticancer agent epirubicin and apoptotic inducer amlodipine were encapsulated into the liposome aqueous core,and the surface of the liposome was modified using dequalinium.The objective of the present study was to establish a high performance liquid chromatography (HPLC) method for the determination of epirubicin,amlodipine and dequalinium in the liposomes.Analysis was performed on an ODS column with an isocratic elution at ambient temperature.Mobile phase was consisted of acetonitrile,0.02 M NaH_2PO_4 and triethylamine(34:66:0.3,v/v/v,pH 4.0).The detection wavelength was set at 240 nm and the flow rate was 1.0 mL/min.The results showed that the calibration curves of epirubicin,amlodipine and dequalinium were linear in the range of(1-50)μg/mL(r^2= 0.9999), respectively.The mean recoveries of epirubicin,amlodipine,and dequalinium were in the range of 95.86%-97.52%,97.17%-98.92% and 98.04%-101.13%,respectively.The contents of epirubicin,amlodipine and dequalinium in the liposomes were in the range of(564.2-606.1)μg/mL,(641.0-704.0)μg/mL,and(816.0-898.0)μg/mL,respectively.The encapsulation efficiencies of epirubicin and amlodipine were around 90%,and the modification rate of dequalinium was approximate 70μg/μmol lipids.The proposed HPLC method was simple and accurate for the simultaneous determination of epirubicin,amlodipine and dequalinium in newly developed anti-resistant liposomes.