Mitochondrial dysfunction is increasingly recognized as a central mechanism by which xenobiotics exert nephrotoxic effects, given the kidney’s high mitochondrial density and energy demand for tubular reabsorption and filtration. This study developed a mitochondrial-targeted lipid nano-formulation co-encapsulating thymoquinone (TQ) and resveratrol (RV) to restore mitochondrial function and prevent Bisphenol S induced mitochondrial dysfunctions in NRK52e cells. Mitochondrial targeting was achieved through conjugation of the SS-31 azido lysine-modified peptide with DSPE-PEG3.4K-DBCO via strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry. Reaction progress was monitored by UV-Visible spectroscopy measuring λmax at predetermined intervals, disappearance of characteristic DBCO absorption peaks at 290 nm and 310 nm confirmed successful conjugation. FTIR spectroscopy showed the reaction completion via elimination of the azide stretch at 2093 cm⁻¹, TIMS-TOF-MALDI-MS revealed product ions at m/z 1374 and 4545, confirming the expected molecular weights of the conjugated species. The resulting peptide-lipid conjugate was incorporated into lipid nanoparticles and characterized for particle size, PDI, and zeta potential which is found to be 81.33±2.13, 0.35±0.04 and -1.97±0.34 mV respectively suitable for mitochondrial delivery. Drug encapsulation efficiency was determined by HPLC analysis and found to be above 95 % for both antioxidants. Biological efficacy was evaluated using MTS cell viability assays, demonstrated that BPS exposure reduced cellular viability to 50% in untreated controls. Remarkably, co-treatment with the mitochondrial-targeted nano-formulation restored cell viability to >90%, indicating significant cytoprotective effects. Mitochondrial functional assays using JC-1, MitoSOX and ICC using antiTOMM20 antibody staining revealed that the formulation effectively preserved mitochondrial membrane potential under BPS-exposed conditions, suggesting successful restoration of mitochondrial bioenergetics. This study demonstrates the successful development of SS-31 peptide-functionalized lipid nanoparticles co-encapsulating thymoquinone and resveratrol as a promising therapeutic strategy for combating xenobiotics-induced mitochondrial dysfunction in renal cells.