Idelalisib (ILB) is a selective phosphatidylinositol-3-kinase delta inhibitor approved for the treatment of hematological malignancies. However, ILB frequently causes hepatotoxicity, and the exact mechanism remains unclear. The current study profiled the metabolites of ILB in mouse liver, urine, and feces. The major metabolites found in the liver were oxidized metabolite GS-563117 (M1) and ILB-glutathione (GSH) adduct (M2). These metabolic pathways were confirmed by analysis of urine and feces from mice treated with ILB. Identification of ILB-GSH adduct (M2) suggests the formation of reactive metabolites of ILB. We also found that M1 can produce reactive metabolites and form M1-GSH adducts. The GSH-conjugates identified in mouse liver were also found in the incubations of ILB and M1 with human liver microsomes. Furthermore, we illustrated that CYP3A4 and 2C9 are the key enzymes contributing to the bioactivation pathway of ILB and M1. In summary, our work revealed that both ILB and its major metabolite M1 can undergo bioactivation to produce reactive metabolites in the liver. Further studies are required to determine whether these metabolic pathways contribute to ILB hepatotoxicity.