Eupatilin, a major pharmacologically active ingredient in StillenTM, has been known to possess anti-peptic, anti-cancer and anti-allergy activities. A rapid, simple, sensitive and specific high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous determination of eupatilin and its main metabolite (eupatilin-7β-O-glucuronide, E-7-G) in rat plasma and tissues was established and validated. The linear range of eupatilin and E-7-G was 0.20˜500 ng/mL and 1.00–2500 ng/mL, and the lowest limit of quantification (LLOQ) of eupatilin and E-7-G was 0.20 and 1.00 ng/mL, respectively. The inter-day and intra-day precision of this assay was restricted to within 10%, with a highest accuracy of more than 90%. The matrix effect, recovery and stability of both eupatilin and E-7-G were all demonstrated to be within acceptable limits. The validated method was then successfully applied to a pharmacokinetics and tissue distribution study. The absolute bioavailability (F) of eupatilin was estimated to be 2.7%. After intravenous administration, eupatilin was degraded with high clearance (14.82 L/kg/h) and a short half-life t1/2 (0.29 h). Eupatilin was rapidly metabolized to E-7-G with systemic exposure at 1288.8 ng h ml-1, while the levels of the latter declined more slowly, with a longer t1/2 (4.15 h). Moreover, both eupatilin and E-7-G were widely distributed across various tissues, including the liver, kidney and intestine. Taken together, eupatilin showed poor absorption, extensive metabolism into E-7-G and a wide tissue distribution, especially in the intestine. These pharmacokinetic results yield helpful insights into the pharmacological actions of eupatilin.