Pulmonary delivery of nucleic acids opens the possibility for direct treatment of lung diseases, like fibrosis, cancer, and infections. Lung retention and biodistribution of nucleic acids remain important issues for the development of suitable therapeutic approaches. Moreover, monitoring the dynamic biodistribution processes of siRNA after aerosol delivery can help in identifying bottlenecks and optimizing therapeutic concepts. We investigated dynamic biodistribution events after intratracheal application of chemically stabilized siRNA labelled with near infrared emitting dye AlexaFluor750 (AF750). Epifluorescence imaging was combined with spectral unmixing to improve the signal to noise ratio. Transillumination imaging has been utilized for quantitative fluorescence imaging tomography (FLIT) together with contrast agent enhanced X-ray absorption computed tomography (CT). Spectral unmixing allowed unambiguous detection of AF750 signals, which could be clearly distinguished from food derived autofluorescence. After successful delivery to the lung, fluorescent signals were also observed in kidneys and bladder, indicating renal excretion of AF750-siRNA. Gel electrophoresis of urine samples showed presence of intact siRNA, at least to a considerable extent. FLIT/CT allowed signal quantification and precise allocation to anatomical structures.