Kidney injury molecule-1 (Kim-1) has been qualified by the Food and Drug Administration and European Medicines Agency as a highly sensitive and specific urinary biomarker to monitor drug-induced kidney injury in preclinical studies and on a case-by-case basis in clinical trials. Here we report the development and evaluation of a rapid direct immunochromatographic lateral flow 15-min assay for detection of urinary Kim-1 (rat) or KIM-1 (human). The urinary Kim-1 band intensity using the rat Kim-1 dipstick significantly correlated with levels of Kim-1 as measured by a microbead-based assay, histopathological damage, and immunohistochemical assessment of renal Kim-1 in a dose- and time-dependent manner. Kim-1 was detected following kidney injury induced in rats by cadmium, gentamicin, or bilateral renal ischemia/reperfusion. In humans, the urinary KIM-1 band intensity was significantly greater in urine from patients with acute kidney injury than in urine from healthy volunteers. The KIM-1 dipstick also enabled temporal evaluation of kidney injury and recovery in two patients who developed postoperative acute kidney injury following cytoreductive surgery for malignant mesothelioma with intraoperative local cisplatin administration. We hope that future, more extensive studies will confirm the utility of these results, which show that the Kim-1/KIM-1 dipsticks can provide a sensitive and accurate detection of Kim-1/KIM-1, thereby providing a rapid diagnostic assay for kidney damage and facilitating the rapid and early detection of kidney injury in preclinical and clinical studies.