This study aimed to investigate the impact of administration routes in establishing the Adriamycin (ADR)-induced chronic kidney disease (CKD) model. Using BALB/c mice, we compared the effects of conventional tail-vein injection (TV10, 10 mg/kg) to those of retro-orbital sinus (orbital vein) injection (OV10, 10 mg/kg
OV8, 8 mg/kg). The results indicated that the OV10 group exhibited CKD pathology similar to the TV10 group, with both groups demonstrating significantly higher urinary albumin/creatinine ratio (p <
0.05), tubular injury (p <
0.05), and degree of renal fibrosis (p <
0.05) than the OV8 group. No significant differences were observed between the OV10 and TV10 groups in urinary albumin/creatinine ratio, tubular injury, and degree of renal fibrosis. These findings demonstrated that retro-orbital administration of 10 mg/kg ADR induces comparable effects to conventional tail-vein administration. This technique's technical simplicity may improve experimental efficiency, reproducibility, and animal welfare in CKD research. In conclusion, this study validates the utility of retro-orbital injection in CKD model establishment, demonstrating its potential to standardize and improve the reliability of future CKD research protocols.