A sensitive, specific, selective and rapid LC-ESI-MS/MS method has been 
developed and validated for the quantification of defactinib in mice plasma 
using 13C3,15N-tofacitinib as an internal standard (I.S.). Sample preparation 
was accomplished through a liquid-liquid extraction process. Baseline 
chromatographic resolution of defactinib and the I.S. was achieved on an 
Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid 
in water and acetonitrile (25:75, v/v) delivered at a flow rate of 0.5mL/min. 
Defactinib and the I.S. eluted at ∼1.59 and 0.99min, respectively. The total 
chromatographic run time was 2.50min. A linear response function was established 
in the concentration range of 0.13-106 ng/mL. Method validation was performed as 
per regulatory guidelines and the results met the acceptance criteria. The 
intra- and inter-day accuracy and precision were in the range of 5.57-13.3 and 
8.63-12.1%, respectively. Defactinib was found to be stable under various 
stability conditions. This novel method has been applied to a pharmacokinetic 
study in mice.