Acetaminophen (APAP) overdose can cause acute kidney injury (AKI), but its molecular mechanisms remain poorly understood and no effective treatments are currently available. In this study, we performed an integrated analysis combining transcriptomic, proteomic, and phosphoproteomic profiling of kidney tissues from APAP-treated mice to investigate the underlying molecular mechanisms and identify potential therapeutic targets. Ten-week-old male C57BL/6 mice were fasted overnight for 16 hours prior to APAP administration. AKI was induced by intraperitoneal injection of a high dose of APAP (300 mg/kg body weight) for 6 hours (n = 4), while control mice received an equivalent volume of PBS via intraperitoneal injection (n = 4). Kidney tissues were collected from both APAP-treated and control groups for downstream omics analyses.