Objective The simultaneous determination of atractylenolide I, II, III in rat plasma and its pharmacokinetic parameters study using UPLC-MS/MS. Methods The plasma samples were analyzed by UPLC-MS/MS after pretreatment with paracetamol as the internal standard. A reliable ESI source, positive ion mode scanning, and multiple reaction monitoring(MRM) method were developed for the simultaneous determination of atractylenolide I, II, III and chromatography was carried out on a Waters ACQUITY UPLC CSH C18(2.1 mm × 100 mm, 1.7 μm) using a gradient mobile phase consisted of 0.05% phosphoric acid water and acetonitrile at a flow rate of 0.2 mL/min. Calculations of the pharmacokinetic data were performed by using DAS 2.1 software. Results The methodology validation of atractylenolide I, II, III was good, and the validated method was successfully applied to the pharmacokinetic study after the rat oral administration of Atractylodes macrocephala Koidz. Decoction. Conclusion This method was available for pharmacokinetic parameters study after oral administration of Atractylodes macrocephala Koidz. and possibly provided a meaningful basis for clinical fair use of Atractylodes macrocephala Koidz.