Protoapigenone, a unique class of natural flavonoid, exerted significant cytotoxic and antitumoral activities. A simple, sensitive, and rapid LC-MS/MS method for the quantification of protoapigenone in plasma was developed and validated to obtain the pharmacokinetic profile of protoapigenone in beagle dogs. Ethyl acetate-10% ammonium hydroxide (2:1, v/v) was employed for liquid-liquid extraction (recovery > 90%). Under optimal conditions, chromatographic resolution was achieved on a C-18 column using acetonitrile and 10 mM ammonium formate in water (9:1, v/v), as mobile phase at a flow rate of 0.5 ml/min. The established method presented good linearity, selectivity, accuracy, and precision in the range of 0.2-100 ng/ml. Finally, this method was successfully applied to a pharmacokinetic study in beagle dogs following oral administration of protoapigenone.