Ritonavir-boosted protease inhibitor regimens are widely used for HIV chemotherapy. However, ritonavir causes multiple side effects, and the mechanisms are not fully understood. The current study was designed to explore the metabolic pathways of ritonavir that may be related to its toxicity. Metabolomic analysis screened out 26 ritonavir metabolites in mice, and half of them are novel. These novel ritonavir metabolites include two glycine conjugated, two N-acetylcysteine conjugated, and three ring-open products. Accompanied with the generation of ritonavir ring-open metabolites, the formation of methanethioamide and 2-methylpropanethioamide were expected. Upon the basis of the structures of these novel metabolites, five bioactivation pathways are proposed, which may be associated with sulfation and epoxidation. By using Cyp3a-null mice, we confirmed that CYP3A is involved in four pathways of RTV bioactivation. In addition, all these five bioactivation pathways were recapitulated in the incubation of ritonavir in human liver microsomes. Further studies are suggested to determine the role of CYP3A and these bioactivation pathways in ritonavir toxicity.