Apigenin trimethyl ether (5,7,4′-trimethoxyflavone, ATE) is a naturally occurring polymethoxyflavone with a wide range of health-promoting activities. In this study, a sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the quantification of ATE in rat plasma. Protein precipitation was applied as plasma clean-up procedure; the electrospray ionization was operated in its positive ion mode while ATE and formononetin (internal standard) were measured by multiple reactions monitoring (ATE: m/z 313.1 → 298.1; formononetin: 269.2 → 213.3). This LC–MS/MS method displayed good selectivity, sensitivity (lower limit of quantification = 2.5 ng/ml), accuracy (both intra- and inter-day analytical recovery within 100 ± 10%) and precision (both intra- and inter-day RSD <10%). The matrix effect was found to be insignificant. The pharmacokinetic profiles of ATE were subsequently examined in Sprague-Dawley rats after single oral administration (10 mg/kg). When given in an aqueous suspension, ATE was slowly absorbed with quite low plasma exposure (AUC). Fasting further attenuated its oral absorption and led to ˜70% drops in average maximal plasma concentration (Cmax) and AUC. When dosed in a solution formulated with 2-hydroxypropyl-β-cyclodextrin, the oral absorption of ATE was substantially improved with ˜500% increases in average Cmax and AUC. Clearly, aqueous solubility has been identified as a barrier to the oral absorption of ATE. The information obtained from this study will facilitate further medicinal exploration on ATE.