In this work, we established a simple, sensitive and rapid method for quantitative detection of dendrobine levels in plasma using ultra fast liquid chromatography with tandem mass spectrometry (UFLC-MS/MS). The plasma samples were performed by protein precipitation and then the supernatant was extracted by chloroform. An aliquot of extracted residues was injected into UFLC-MS/MS system with separation performed by an ACQUITY HSS T3 column. Acetonitrile and water containing 10 mM ammonium acetate was used as a mobile phase. The average extraction recoveries were greater than 80.5% for dendrobine and 90.9% for matrine (Internal Standards, IS) from plasma. Good linearity (R² > 0.992) was observed in the range of 1–1000 ng mL⁻¹. For rat plasma, the overall accuracy of this method was 94.3–110.3%. Intra-assay and inter-assay precision was less than 11.7%. The lower limit of quantification and detection for dendrobine was 1.0 and 0.4 ng mL⁻¹, respectively. The practical utility of this new UFLC-MS/MS method was confirmed by pharmacokinetic studies followed by sublingual intravenous administration of three levels of dendrobine in rats.