Daphnane diterpenes with a 5/7/6-tricyclic ring system exhibit potent anti-HIV activity but are found in low abundance as plant natural products. In this study, an effective approach based on mass spectrometric fragmentation pathways was conducted to specifically recognize and isolate anti-HIV compounds of this type from <i>Daphne genkwa</i>. Briefly, the fragmentation pathways of reference analogues were elucidated based on characteristic ion fragments of <i>m</i>/<i>z</i> 323 → 295 → 267 or <i>m</i>/<i>z</i> 253 → 238 → 197 by ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS<sup><i>n</i></sup>) and then applied to the differentiations of substances with or without an oxygenated group at C-12. Twenty-seven daphnane diterpenes were successfully recognized from a petroleum ether extract of <i>D. genkwa</i>, including some potential new compounds and isomers that could not be identified accurately only from the ion fragments. Further separation of these target compounds using high-speed countercurrent chromatography (HSCCC) and preparative HPLC led to the isolation of three new (<b>11</b>, <b>25</b>, and <b>27</b>) and 14 known compounds, whose structures were identified and confirmed based on MS, NMR, and electronic circular dichroism (ECD) spectroscopy. The isolates exhibited anti-HIV activities at nanomolar concentrations. The results demonstrated that this strategy is feasible and reliable to rapidly recognize and isolate daphnane diterpenes from <i>D. genkwa</i>.