The small molecule simufilam, also called PTI-125, has been reported to reduce cell overgrowth and seizure activity in a mouse model of tuberous sclerosis complex (TSC). TSC is a multisystem developmental disorder caused by mutations in TSC1 or TSC2, resulting in mTOR complex 1 (mTORC1) hyperactivity and downstream cellular changes that contribute to focal malformations of the developing cortex and abnormal growth throughout the body. Here, to better understand the role of simufilam at the cellular level, we investigated the impact of simufilam on the proteome of TSC2 knockout (KO) HEK293T cells in vitro. We first validated that TSC2 KO cells displayed increased mTORC1 activity and elevated expression of the stress-associated marker ATF4 compared with TSC2 wild-type (WT) cells. Simufilam reduced ATF4 expression but did not attenuate mTORC1 activity. Next, using LC-MS/MS in TSC2 KO cells, we identified 514 differentially abundant proteins following simufilam treatment. Pathway and protein–protein interaction analyses revealed coordinated changes in mitochondrial translation and bioenergetic pathways, including mitochondrial ribosomal proteins and respiratory-chain-associated proteins, as well as pathways related to preribosome biogenesis, rRNA processing, RNA metabolism, and to some extent RHO GTPase-actin signaling, and chromatin/transcriptional regulation. Selected proteins from these pathways, including MRPS30, MRPL43, MRPL16, ALDH1L2, NDUFA6, and RBM19, were further examined by immunoblotting and found to be altered by simufilam treatment. Functionally, simufilam restored mitochondrial membrane potential in TSC2 KO cells. Overall, these findings support a potential mTORC1-independent role for simufilam in attenuating cellular stress and regulating mitochondrial function in TSC.