The rapid diagnosis and quantification of acute kidney injury (AKI) severity remain high clinical priorities. By combining intravital fluorescent ratiometric two-photon kidney imaging and the two-compartment pharmacokinetics model, we demonstrate that rapid quantification of glomerular filtration rate (GFR) can be achieved in physiologic and AKI rat kidney models. Using a bolus infusion of a mixture of FITC-inulin and a 500-kDa Texas Red dextran, a full spectrum of GFR values, ranging from 0.17 to 1.12 ml·min(-1)·100 g(-1), was obtained. The GFR values thus determined correlated well with values obtained by the standard 2-h inulin infusion clearance method with a Pearson's correlation coefficient of 0.85. In addition, postischemia deterioration was studied by measuring GFR using the two-photon approach during 24 h following a 45-min bilateral ischemia clamp model. The GFR was found to decline sharply during the initial 4 h followed by a nadir with little sign of rising over the ensuing 24-h period. Moreover, a FITC-labeled 5-kDa dextran was identified as having nearly identical filtration characteristics as FITC-inulin, but had markedly increased fluorescent intensity, thus minimizing the quantity needed for individual studies. The technique reported allows for very rapid GFR determinations, within 10-15 min, based on plasma clearance of a freely filtered fluorescence probe, instead of a prolonged one-compartment interstitial space reporter molecule clearance employed by other technologies.