TM-2 known as a potential antitumor drug is a novel semi-synthetic taxane derivative. As drug–protein interactions contribute to insights into pharmacokinetic and pharmacodynamic properties, we elucidated the binding of TM-2 to plasma protein. In this study, a simple, rapid and reliable method was developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography–tandem mass spectrometry(UPLC–MS/MS) for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 °C. After liquid–liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLCsC18(2.1 mm 50 mm, 1.7 mm), and acquisition of mass spectrometric data was performed in multiple reaction monitoring(MRM) mode via positive electrospray ionization. The assay was linear over the concentration rang of 5–2000 ng/m L. The intra- and inter-day precisions were 0.1%–14.8%, and the accuracy was from 6.4% to 7.0%. This assay has been successfully applied to a protein binding study of TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4% 7 6.5%(rat),87.9% 7 3.6%(human) and 79.4% 7 4.0%(beagle dog). The results revealed that there was an insignificant difference among the three species. As-protein known as a novel semi-synthetic taxane derivative. As this drug-protein interactions contribute insights into pharmacokinetic and pharmacodynamic properties, we elucidated the binding of TM-2 to plasma protein. In this study, a simple , rapid and reliable method employed developed and validated employing equilibrium dialysis for the separation of bound and unbound drugs and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) for the quantitation. Protein binding reached equilibrium within 24 h of incubation at 37 ° C. After liquid-liquid extraction with methyl tert-butyl ether, the samples were separated on Thermo Syncronis UPLCs C18 (2.1 mm 50 mm, 1.7 mm), and acquisition of mass spectrometric data was performed in multiple reaction monitoring (MRM) mode via positive electrospray ionization. The assay was linear over the concentration rang of 5-2000 ng / m L. The intra- and inter-day precisions were 0.1% -14.8%, and the accurac y was from 6.4% to 7.0%. This assay has been successfully applied to a protein binding study TM-2 in rat, human and beagle dog plasma. TM-2 showed high protein binding of 81.4% 7 6.5% (rat), 87.9% 7 3.6% (human) and 79.4% 7 4.0% (beagle dog). The results revealed that there was an insignificant difference among the three species.