In the current work, a rapid, specific, sensitive and validated liquid chromatography tandem mass-spectrometric method was developed for the quantification of ponatinib (PNT) in human plasma and rat liver microsomes (RLMs) with its application to metabolic stability. Chromatographic separation of PNT and vandetanib (IS) were accomplished on Agilent eclipse plus C18 analytical column (50 mm × 2.1 mm, 1.8 μm particle size) maintained at 21±2°C. Flow rate was 0.25 mLmin-1 with run time of 4 min. Mobile phase consisted of solvent A (10 mM ammonium formate, pH adjusted to 4.1 with formic acid) and solvent B (acetonitrile). Ions were generated by electrospray (ESI) and multiple reaction monitoring (MRM) was used as basis for quantification. The results revealed a linear calibration curve in the range of 5-400 ngmL-1 (r2 ≥ 0.9998) with lower limit of quantification (LOQ) and lower limit of detection (LOD) of 4.66 and 1.53 ngmL-1 in plasma, 4.19 and 1.38 ngmL-1 in RLMs. The intra- and inter-day precision and accuracy in plasma ranged from1.06 to 2.54% and -1.48 to -0.17, respectively. Whereas in RLMs ranged from 0.97 to 2.31% and -1.65 to -0.3%. The developed procedure was applied for quantification of PNT in human plasma and RLMs for study metabolic stability of PNT. PNT disappeared rapidly in the 1st 10 minutes of RLM incubation and the disappearance plateaued out for the rest of the incubation. In vitro half-life (t1/2) was 6.26 min and intrinsic clearance (CLin) was 15.182± 0.477.