Pulmonary formulations have been attracting much attention because of their direct effects on respiratory diseases, but also their non-invasive administration for the treatment of systemic diseases. When developing such formulations, they are typically first investigated in mice. As there are various pulmonary administration methods, the researcher has to decide on the best quantitative method for their preclinical investigations among candidate methods, both for total delivery and distribution within the lung lobes. In this study, we investigated the deposition and distribution of siRNA loaded PLGA nanoparticles (NPs) in the different lung lobes via three widely used pulmonary administration methods: intratracheal instillation, intratracheal spraying and intranasal instillation. The NPs were radiolabeled with 111In, administered and a single photon emission computed tomography (SPECT/CT) whole body scan performed. Quantitative image volume of interest (VOI) analysis of all inhalation related organs was performed, plus sub-organ examinations using dissection and gamma counting. Intratracheal instillation and intratracheal spraying deposited >95% and >85% of radiolabeled NPs in the lung, respectively. However, the lung lobe distribution of the NPs was inhomogeneous. Intranasal instillation deposited only ∼28% of the dose in the lungs, with even larger inhomogeneity and individual variation between animals. Furthermore, there was a high deposition of the NPs in the stomach. Intratracheal instillation and intratracheal spraying deposit a large number of NPs in the lungs, and are thus useful to test therapeutic effects in preclinical animal studies. However, the inhomogeneous distribution of formulation between lung lobes needs to be considered in the experimental design. Intranasal instillation should not be used as a means of pulmonary administration.