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Fuzi (lateral root of Aconitum carmichaelii) has been used for millennia in Traditional Chinese Medicine (TCM) and Ayurveda to treat cancer and cardiovascular diseases. Fuzi must be processed before use to decrease the concentration of its toxic alkaloids. Detoxification during processing occurs through the transformation of diester-diterpenoid alkaloids (DDAs) to monoester-diterpenoid alkaloids (MDAs). However, traditional detoxification methods are time-consuming and expensive on large-scale production. To develop efficient detoxification protocols to reduce unnecessary processing procedure and keep the maximum functional contents from raw Fuzi, we replicated the traditional procedure and quantified the DDAs and MDAs by UPLC-MS/MS and UPLC-PDA during different steps and conditions of processing. With due consideration of obtained data, we concluded that soaking in Danba solution and the washing steps were inefficient traditional processing methods. The detoxification effect of steaming (56.3 ± 0.27 μg/g DDAs, lowest after steaming) was weaker and slower than boiling (5.8 ± 0.33 μg/g DDAs, lowest after boiling). Moreover, roasting at 105℃ showed better effect in lowering the DDAs (5.8 ± 0.33 μg/g DDAs) and increasing the MDAs (729.1 ± 1.22 μg/g MDAs, highest) than roasting at 60℃ (17.3 ± 0.65 μg/g DDAs; 504.0 ± 0.99 μg/g MDAs). With these highly reliable analytic data, we established an efficient and referenceable detoxification protocol for Fuzi TCM products, in which DDAs and MDAs should legitimately follow the safe and specific ranges stipulated in pharmacopeias.