A new separation method, negative pressure cavitation (NPC) extraction followed by HPLC-MS-MS for the determination of paclitaxel and its analogues in the needles of Taxus species is described in this study. Compared with three conventional extraction methods, NPC is a more effective, economical, and facile method for the separation of nature compounds from herbal plants. In contrast to high-temperature extraction, NPC at low temperature can minimize undesirable reactions such as thermal decomposition or degradation. In addition, the extraction mechanism of NPC was also illuminated. The effects of vacuum degree, extraction solvent, ratio of solid to liquid, time, and times extraction were studied and optimized. The optimum extraction conditions were as follows: vacuum degree -0.03 MPa, solvent 80% v/v alcohol, ratio of material to liquid 1:15 (g/mL), extraction time 60 min, extraction (x3). Using these conditions, the recoveries of 10-deacetyl-7-xylosylpaclitaxel, 10-deacetylpaclitaxel, cephalomannine, and paclitaxel were higher than 95.88, 95.82, 94.85, and 96.18%, respectively. The contents of paclitaxel, 10-deacetyl-7-xylosylpaclitaxel, 10-deacetylpaclitaxel, and cephalomannine for Taxus chinensis were 0.0053, 0.0467, 0.0132, and 0.0076%, and 0.0067, 0.0153, 0.0047, and 0.0064% for Taxus cuspidata, respectively. Furthermore, a comparison of SEM images observed the morphological changes of microstructures and cellular damage in yew needles.