L-Isoleucine has no absorption in the ultraviolet ( UV) region and needs to be derivatized before determination by UV detector． The aim of this work is to compare the two pre-column derivatization methods: OPA automatic derivatization and PITC manual derivatization，and establish an efficient method for the determination of L-isoleucine content by high performance liquid chromatography． The total detection time with OPA automatic derivatization method took 15 minutes with Ｒ-value of 1，and the recoveries were from 99% to 100． 1% with ＲSD ＜ 1%． The PITC manual derivatization took 30 min， with the recovery rate of 96% to 98% ( ＲSD ＜ 1%) ． OPA automatic derivatization exhibited obvious advantages in the detection timeliness and accuracy，which can be used to efficiently detect L-isoleucine content． It has high application value in microbial fermentation，feed additives，food additives and pharmaceuticals．