This study aimed to develop potent and selective PI3Kα inhibitors using molecular hybridization and investigate their antitumor activity and mechanism. Sixteen quinazoline-2-indolinone derivatives were synthesized and evaluated for their inhibitory activity against PI3Kα and selectivity towards PI3Kβ, γ, and δ. Compound 8, with a bis-benzyl substitution, exhibited the best inhibitory activity against PI3Kα (IC50 = 9.11 nM) and showed 10.41, 16.99, and 37.53-fold higher selectivity towards PI3Kα over PI3Kβ, γ, and δ, respectively. In vitro and in vivo experiments demonstrated that compound 8 significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) cells, induced mitochondrial dysfunction, and activated the mitochondrial apoptotic pathway. Compound 8 also suppressed the PI3K/Akt/mTOR pathway in NSCLC cells, suggesting its potential as a novel therapeutic agent for NSCLC. In vivo studies in a mouse model of NSCLC showed that compound 8 reduced tumor weight and volume, and induced apoptosis without significant toxicity to major organs. These findings highlight the potential of compound 8 as a promising PI3Kα inhibitor for the treatment of NSCLC.This study aimed to develop potent and selective PI3Kα inhibitors using molecular hybridization and investigate their antitumor activity and mechanism. Sixteen quinazoline-2-indolinone derivatives were synthesized and evaluated for their inhibitory activity against PI3Kα and selectivity towards PI3Kβ, γ, and δ. Compound 8, with a bis-benzyl substitution, exhibited the best inhibitory activity against PI3Kα (IC50 = 9.11 nM) and showed 10.41, 16.99, and 37.53-fold higher selectivity towards PI3Kα over PI3Kβ, γ, and δ, respectively. In vitro and in vivo experiments demonstrated that compound 8 significantly inhibited the proliferation and migration of non-small cell lung cancer (NSCLC) cells, induced mitochondrial dysfunction, and activated the mitochondrial apoptotic pathway. Compound 8 also suppressed the PI3K/Akt/mTOR pathway in NSCLC cells, suggesting its potential as a novel therapeutic agent for NSCLC. In vivo studies in a mouse model of NSCLC showed that compound 8 reduced tumor weight and volume, and induced apoptosis without significant toxicity to major organs. These findings highlight the potential of compound 8 as a promising PI3Kα inhibitor for the treatment of NSCLC.