Taxane compounds have attracted wide attention due to the basic chemical structure of taxol as an alternative anticancer drug. The full-scan tandem mass spectrometry (MS/MS) fragmentation behaviors of seven taxane compounds were studied. For taxanes of Sc-T and Sc-T-Xyl types, diagnostic product ions are originated from a cleavage in the ester bond of the C 13 position and the C−O bond of the C 7 position, and the subsequent fragmentation pattern is similar to those of M-type taxanes with the loss of different numbers of acetic acid moieties (AcOH), benzoic acid moieties (BzOH), and H 2 O molecules. A rapid (7 min) and one-step screening method of two-dimensional microscale carbon fiber and active carbon fiber columns combined with tandem mass spectrometry (2DμCFs-MS/MS) was developed for the screening of taxane compounds from Taxus cuspidata samples. Before MS/MS analysis, the 2DμCFs system can group the sample extract without any pretreatment into three chromatographic-type fractions of strong, medium, and weak polarity to avoid matrix interference, such as lipids and pigments. The 2DμCFs-MS/MS can also conduct qualitative and quantitative analysis of taxane compounds, which is evaluated by limits of detection ranging from 3 to 50 ng mL −1 , limits of quantitation ranging from 10 to 150 ng mL −1 , satisfactory recoveries from 75.2 to 112.2%, and reproducibilities with relative standard deviations from 1.4 to 11.7%.