Abstract: Objective To develop a simple,precise and accurate high performance liquid chromatographic method for quantitative determination of acid sphingomyelinase activity in biological samples.Methods The biological samples of blood,urine and liver homogenate were collected from adult Kunming mice and incubated with fluorescent substrate BODIPY C₁₂-Spm for 2 hours.The samples were detected using ACQUITY BEH C18 chromatographic column with mobile phase containing methanol and 13 mM sodium acetate(95:5,v/v)at a flow rate of 0.8 ml/min,with the injection volume of 10 μL.The eluent was monitored with a fluorescence detector set to excitation and emission wavelengths of 505 nm and 540 nm,respectively.Results Fluorescent product B₁₂-Cer in reverse analytical chromatography was effectively separated in 4 min,and the linearity of the method was good in a range of 40-5 000 nm,with a correlation coefficient of 0.9988.The detection limit of the method was 5.86 nmol/L.The mean of acid sphingomyelinase activity in blood,urine and liver homogenate of the mice was 927.40±189.62,122.88±49.84,and 422.51±65.81 nmol/μl/h,respectively.Conclusion The high performance liquid chromatographic method based on fluorescent substrate is a simple,stable and high sensitive technique to determine the activity of acidic pahospholipase in trace biological samples effectively.