Understanding the uptake and clearance kinetics of new drugs and contrast agents is an important aspect of drug development that typically involves a combination of imaging and analysis of harvested organs. Although these techniques are well-established and can be quantitative, they generally do not preserve high resolution biodistribution information. In this context, fluorescence whole-body cryo-imaging is a promising technique for recovering 3D drug/agent biodistributions at a high resolution throughout an entire study animal at specific time points. A common challenge associated with fluorescence imaging in tissue is that agent signal can be confounded by endogenous fluorescence signal which is often observed in the visible window. One method to address this issue is to acquire hyperspectral images and spectrally unmix agent signal from confounding autofluorescence signals using known spectral bases. Herein, we apply hyperspectral whole-body cryo-imaging and spectral unmixing to examine the distribution of multiple fluorescent agents in excretion organ regions.