BJECTIVE: To develop a determination method of etoposide phosphate and related substance. METHODS: A μ Bondpak^TM Phenyl Column (3.9mm×300mm,10m,Waters) was used.The mobile phase consisted of acetonitrile-0.02mol·L^-1 ammonium dihydrogen phosphate solution (22:78) with the pH adjusted to 2.5 with phosphoric acid.Detection was performed with UV detector at 230nm. RESULTS: The calibration curve for etoposide phosphate was linear within the concentration range of 0.04920～0.4428mg·ml^-1(r=0.9999,n=5).The average recovery was 99.8%.The limits of detection for etoposide phosphate,etoposide and lignan P phosphate were 2.5,2.4 and 0.45ng,respectively. CONCLUSION: The method had the advantages of simplicity,rapidity,accuracy and precision.