In this research, the simultaneous determination of paclitaxel and baccatin III, using HPLC was developed and validated. The chromatographic condition included Luna L43 column (250 x 4.6 mm, 5 um), the mobile phase: acetonitrile and water in the gradient process. Detective wavelength was 228 nm. This method was validated on the specificity, accuracy and repeatability. The developed method was applied for the quantitation of paclitaxel and baccatin III in young branch of T. wallichiana. Results showed that the content of paclitaxel and baccatin III were 0.0314 +/- 0.0006 percent and 0.0540 +/- 0.0003 percent, respectively.