Abstract: Effect of pre-column derivatization on determination of artemisinin from Artemisia annua by HPLC-UV was studied. The modified determinatin method of HPLC-UV with pre-column derivatization was established as follows:the standard solution of artemisinin (or its extract) was supplementally added to 15.0mL with 95% ethanol, then added with 0.2% NaOH 8.0mL, andheated in a water bath at 45℃ for 30min. After it was cooled to room temperature, acidified with 0.08mol/L HAc 10mL and shaked. Then it was filled to marked volume of 50 mL with 95% ethanol, separated by HPLC, and detected at 260nm. The methodgave a wide range of linearity in the concentration range from 0 to 200mg/L. The relative standard deviation(RSD) was 1.40% and average recovery(n=5) was 100.1%. The method was used to determine artemisinin extracted from introduced sample. The result demonstrated that the RSD of stabilization and reproducibility were 4.35% and 4.96%, respectively. It fully proved that the method is simple, stable and reproducible.