Potentially transplantable kidneys experience warm ischemia, and this injury is difficult to quantify. We investigate optical spectroscopic methods for evaluating, in real time, warm ischemic kidney injury and reperfusion. Vascular pedicles of rat kidneys are clamped unilaterally for 18 or 85 min, followed by 18 or 35 min of reperfusion, respectively. Contralateral, uninjured kidneys serve as controls. Autofluorescence and cross-polarized light scattering images are acquired every 15 susing 335-nm laser excitation (autofluorescence) and 650±20-nm linearly polarized illumination (light scattering). We analyze changes of injured-to-normal kidney autofluorescence intensity ratios during ischemia and reperfusion phases. The effect of excitation with 260 nm is also explored. Average injured-to-normal intensity ratios under 335-nm excitation decrease from 1.0 to 0.78 at 18 min of ischemia, with a return to baseline during 18 min of reperfusion. However, during 85 min of warm ischemia, average intensity ratios level off at 0.65 after 50 min, with no significant change during 35 min of reperfusion. 260-nm excitation results in no autofluorescence changes with ischemia. Cross-polarized light scattering images at 650 nm suggest that changes in hemoglobin absorption are not related to observed temporal behavior of the autofluorescence signal. Real-time detection of kidney tissue changes associated with warm ischemia and reperfusion using laser spectroscopy is feasible. Normalizing autofluorescence changes under 335 nm using the autofluorescence measured under 260-nm excitation may eliminate the need for a control kidney.