Abstract: This paper described a rapid and sensitive HPLC method to analyze (E)-3,5,4′-trimethoxystilbene (BTM-0512) in rat plasma and tissues. The analysis used a BDS Hypersil C₁₈ analytical column (250 mm×4.6 mm ID, 5 μm) and acetonitrile / water as the mobile phase. The UV detection wavelength was 319 nm. Proteins were precipitated with acetonitrile and diethylstilbestrol as internal standard. The method was validated according to State Food and Drug Administration of China and ICH of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines. The limit of detection (S/N: 3/1) for BTM-0512 was 0.005 μg·mL^-1 for plasma. The method performances were shown to be selective for BTM-0512 and the linearity of the assay method was up to 10.0 μg·mL^-1 and 40.0 μg·g^-1 for plasma and tissues, respectively. At 0.1, 1 and 5 μg·mL^-1 (n=5), intraday and interday precision values (%RSD) were in the range of 2.6%-5.1% and 2.4%-4.8%, respectively. Mean accuracy and absolute recoveries of BTM-0512 ranged from 95.3%-100.1% and 95.9%-100.9% for plasma and tissues, respectively. This method can be quite useful for BTM-0512 pharmacokinetic and tissue distribution studies, for purpose which multiple plasma and tissue samples can be analyzed quickly with high reproducibility.