Purpose: To develop and validate a fast, sensitive, and simple liquid chromatographic method coupled with tandem mass spectrometry for the determination of the potent tyrosine kinase inhibitor, ninetedanib (NTB) in plasma, utilizing cyclobenzaprine (CBP) as internal standard (IS). Methods: Separation of the two components (NTB and CBP) was performed on a pentafluorophenyl (PFP) reversed phase column (50 x 2 mm, 3 mu m) at ambient temperature using isocratic elution with acetonitrile-water (60:40, v/v) containing 0.01 M ammonium formate buffer (pH 4.2) at a flow rate of 0.4 mL/min. NTB and CBP were monitored by a triple quadrupole tandem mass spectrometer with electrospray ionization source in the positive ion mode. The current method was validated following the European Medicines Agency (EMA) guidelines Results: The proposed method allowed rapid and specific quantification of NTB in the calibration range of 2 - 150 ng/mL and determination coefficient of >= 0.999. Intra- and inter-day accuracy and precision were < 4 % in all cases. Conclusion: The developed procedure is rapid, specific, reliable, and validated for quantification of NTB in human plasma, and thus can be applied efficiently for the analysis of clinical samples containing NTB.