Fluorescence imaging in diffusive media locates tumors tagged by injected fluorescent markers in NIR wave-lengths. For deep embedded markers, natural autofluorescence of tissues comes to be a limiting factor to tumor detection and accurate FDOT reconstructions. A spectroscopic approach coupled with Non-negative Matrix Factorization source separation method is explored to discriminate fluorescence sources according to their fluorescence spectra and remove unwanted autofluorescence. We successfully removed autofluorescence from acquisitions on living mice with a single subcutaneous tumor or two capillary tubes inserted at different depths.© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.