To establish a HPLC method for a new compound 1,3-O-dicaffeoylglycerides determination.The separation was performed in a Kromasil C18 column (4.6 mm x 150 mm, 5 microm) with a mobile phase of 0.1% H3PO4: Acetontrile = 75 : 25 (v/v). The flow rate was 1.0 ml x min(-)1 and the temperature of column was 30 degrees C.A satisfactory separation between 1, 3-O-dicaffeoylglycerides and impurity was obtained. The calibration curve was linear over the concentration range from 0.051 6 microg to 0.516 microg, r = 0.999 9. The average recoveries was 97.1% (RSD 1.3%). The content of 1,3-O-dicaffeoylglycerides in pineapple leaves from three different batches were 0.033%, 0.034% and 0.031% respectively.The method has good selectivity, high recovery and reproducibility, and can be used for the analysis of 1,3-O-dicaffeoylglycerides in pineapple leaves and their quality control.