Sensitive kidney safety assessment is important for successful drug development in both preclinical and clinical stages. The Food and Drug Administration recently qualified a composite measure of 6 urine creatinine-normalized biomarkers, such as clusterin, cystatin C, kidney injury molecule 1 (KIM-1), N-acetyl-β-d-glucosaminidase, neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin, for monitoring kidney toxicity in early clinical trials. The qualification was based on small molecule drugs in humans, and the full panel has not been assessed in other species or for other drug modalities. This study evaluated the effects on these biomarkers for a constrained ethyl antisense oligonucleotide (tool ASO) with demonstrated kidney toxicity in mice compared to a control ASO of the same chemistry. Dosing 50 mg/kg of the tool ASO resulted in mild proximal tubular pathology and elevations in KIM-1, clusterin, NGAL, and cystatin C. A lower dose resulted in milder histopathology and lower biomarker increases. Unexpectedly, the control ASO induced mild elevations in KIM-1, NGAL, and cystatin C, despite the lack of pathology. Both KIM-1 and clusterin were most closely associated with kidney pathology and increased with the severity of injury. Altogether, our data suggest that a biomarker panel is a sensitive tool for the detection of preclinical ASO-induced kidney pathology.