Abstract: A method of RP-HPLC with a ODS-C₁₈column and a UV detector was developed to separate and detect three metabolites such as the caffic acid (CA), 4-conmaric acid (PA) and ferulic acid (FA), in the biosynthetic pathway of tobacco lignin.The opti mum separation conditions were suggested with the mobile phase containing 1.5% acetic acid (V (methanol):V(water)=20:80) and flowrate 1.0mL/min and the UV detection wavelength of 320nm.The recovery rates of CA,PA and FA were 95.59%,93.67%and 97.32%, and the linear correlation coefficients were 0.9946,0.9946and 0.9945 respectively.The results showed that the method of RP-HPLC was suitable for quantitative analysis of the three metabolites.The contents of CA,PA and FAin transgenic tobacco plants were 2.6-3.0,3.4-4.1 and 7.1-8.2 fold higher than those in wild type tobacco plants.