Growing evidence has highlighted that mitochondrial dysfunction contributes to drug-induced toxicities and leads to drug attrition and post-market withdrawals. The acetylation or deacetylation of mitochondrial proteins can affect mitochondrial functions as the cells adapt to various cellular stresses and other metabolic challenges. SIRTs act as critical deacetylases in modulating mitochondrial function in response to drug toxicity, oxidative stress, reactive oxygen species (ROS), and energy metabolism. We previously showed that a recently characterised SIRT inhibitor (BZD9L1) is non-toxic in rodents in a short-term toxicity evaluation. However, the impact of BZD9L1 on mitochondrial function is unknown. This work aims to determine the effects of BZD9L1 on mitochondrial function in human normal liver and kidney-derived cell lines using the Agilent Seahorse Cell Mito Stress Test to complement our short-term toxicity evaluations in vivo. The Mito Stress assay revealed that BZD9L1 could potentially trigger oxidative stress by inducing ROS, which promotes proton leak and reduces coupling efficiency in liver-derived THLE cells. However, the same was not observed in human kidney-derived HEK293 cells. Interestingly, BZD9L1 had no impact on SIRT3 protein expression in both cell lines but affected SOD2 and its acetylated form at 72 h in THLE cells, indicating that BZD9L1 exerted its effect through SIRT3 activity rather than protein expression. In contrast, BZD9L1 reduced SIRT1 protein expression and impacted the p53 protein differently in both cell lines. Although BZD9L1 did not affect the spare respiratory capacity in vitro, these findings call for further validation of mitochondrial function through assessment of other mitochondrial parameters to evaluate the safety of BZD9L1.