Objective To investigate tissue distribution of Hylotelephin in Beagle dogs and excretion of Hylotelephin in rats. Methods A high performance liquid chromatography (HPLC) with UV detection was developed to study the concentrations of Hylotelephin in biological samples, taking anthracene as an internal standard, Benzoyl chloride as the pre-column derivatization of Hylotelephin and methanol-water as the mobile phase. Results After a single intravenous dose of 10.6 mg/kg Hylotelephin in beagle dogs, parent drug was widely distributed to virtually all tissues in 5 min and the concentration of Hylotelephin in most tissues at 90 min were lower than those at 5 min obviously. Hylotelephin was mainly distributed in kidney, liver and spleen, secondly in heart, lung and intestine. The parent drug concentration in kidney, liver and spleen was similar to that in blood at the same time point. After a single intravenous dose of 36 mg/kg Hylotelephin in rats, the excretion of the parent drug in urine, feces and bile amounted to 88.45%, 0.61% and 1.08% of the dose, respectively. The parent drug excretion amounted to 67% of the dose in 3 h. Conclusion Hylotelephin was distributed and eliminated in Beagle dogs rapidly. It was mostly distributed in kidney, liver, spleen and plasma. The parent drug excretion was 88% via urine.