Rhododendri Mollis Flos (R. mole Flos), the dried flowers of Rhododendron mole G. Don, have the ability to relieve pain, dispel wind and dampness, and dissolve blood stasis, but they are highly poisonous. The significance of this study is to explore the analgesic application potential of R. mole Flos and its representative component. According to the selected processing methods recorded in ancient literature, the analgesic activities of wine- and vinegar-processed R. mole Flos, as well as the raw product, were evaluated in a writhing test with acetic acid and a formalin-induced pain test. Subsequently, the HPLC-TOP-MS technique was utilized to investigate the changes in active components before and after processing once the variations in activities were confirmed. Based on the results, rhodojaponin VI (RJ-Vl) was chosen for further study. After processing, especially in vinegar, R. mole Flos did not only maintain the anti-nociception but also showed reduced toxicity, and the chemical composition corresponding to these effects also changed significantly. Further investigation of its representative components revealed that RJ-VI has considerable anti-nociceptive activity, particularly in inflammatory pain (0.3 mg/kg) and peripheral neuropathic pain (0.6 mg/kg). Its toxicity was about three times lower than that of rhodojaponin III, which is another representative component of R. mole Flos. Additionally, RJ-VI mildly inhibits several subtypes of voltage-gated sodium channels (IC > 200 μM) that are associated with pain or cardiotoxicity. In conclusion, the chemical substances and biological effects of R. mole Flos changed significantly before and after processing, and the representative component RJ-VI has the potential to be developed into an effective analgesic.