Atractylodis exerted a variety of pharmacological effects such as anti-tumor, anti-inflammatory, anti-bacterial, and anti-aging effects etc. The major ingredients of Atractylodis are atractylenolide I and II that exhibited activities in anti-inflammatory and anticancer. In this work, a sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method for determination of atractylenolide I and II in rat plasma was developed. The UPLC–MS/MS method was validated for selectivity, linearity, accuracy, precision, recovery, and stability with a total run time of 4.0 min. After addition of atractylenolide III as an internal standard (IS), protein precipitation by acetonitrile was used to prepare samples. Chromatographic separation was achieved on a UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) with 0.1% formic acid and acetonitrile as the mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; multiple reactions monitoring (MRM) mode was used for quantification using target fragment ions m/z 231.1 → 185.1 for atractylenolide I, m/z 233.1 → 91.0 for II, and m/z 249.0 → 231.1 for IS. Calibration plots were linear throughout the range 1–1000 ng/mL for atractylenolide I and II in rat plasma. Mean recoveries of atractylenolide I and II in rat plasma ranged from 86.2% to 96.3%. Relative standard deviation (RSD) of intra-day and inter-day precision was both less than 12%. The accuracy of the method was between 91.0% and 109.0%. The method was successfully applied to pharmacokinetic study of atractylenolide I and II after intravenous administration in rats.