Fluorescent imaging in diffusive media is an emerging imaging modality for medical applications: injected fluorescent markers bind specifically to targeted compounds, like carcinoma. To investigate thick media for medical diagnostic application, as the fluorescence signal gets exponentially weak with the light travel distance, any disturbing signal, such as biological tissues intrinsic fluorescence (IF), may be a limiting factor. We processed the NMF algorithm on the fluorescence acquisition obtained along the laser line on the back of the mouse. We found that in vivo spectrally resolved acquisition combined to NMF processing successfully filters fluorescence contributions of interest from measurements impaired by unwanted signals.