Objective The visual inspection method were not appropriate to perform a hemolysis evaluation for colored injection like doxorubicin hydrochloride,this article adopted three methods to evaluate the hemolysis test of doxorubicin hydrochloride in vitro and provide reference for clinical drug safety.Methods Using rabbit erythrocytes as experimental object,the durg concentration 4.0 and 2.0 mg/mL was chosen which range of clinical concentration and preclinical safety evaluation concentration,to evaluate the hemolysis test of doxorubicin hydrochloride injection with blood analyzer test,direct colorimetric assay,and indirect colorimetric assay.Results The evaluation results of three different methods were very consistent.The tube's hemolysis rate of 4.0 mg/mL dose was far greater than 5％,which means serious hemolysis;Only 0.1 mL tube of 2.0 mg/mL dose (according to the drug concentration equal to 0.5 mL tube of 0.4 mg/mL drug concentration) without hemolysis occurring,the other tubes' hemolysis rates were far greater than 5％,which means serious hemolysis.Conclusion The hemolysis phenomenon may occur when 2.0 mg/mL dose of doxorubicin hydrochloride solution for iv injection is used in clinic and dilution (final concentration not more than 0.4 mg/mL) is recommended.