Respiratory syncytial virus (RSV) is the most common viral cause of bronchiolitis and pneumonia in children twelve months of age or younger and a significant cause of lower respiratory disease in older adults. As various clinical and preclinical candidates advance, cotton rats (Sigmodon hispidus) and non-human primates (NHP) continue to play a valuable role in RSV vaccine development, since both animals are semi-permissive to human RSV (HRSV). However, appropriate utilization of the models is critical to avoid mis-interpretation of the preclinical findings. Using a multimodality imaging approach; a fluorescence based optical imaging technique for the cotton rat and a nuclear medicine based positron emission tomography (PET) imaging technique for monkeys, we demonstrate that many common practices for intranasal immunization in both species result in inoculum delivery to the lower respiratory tract, which can result in poor translation of outcomes from the preclinical to the clinical setting. Using these technologies we define a method to limit the distribution of intranasally administered vaccines solely to the upper airway of each species, which includes volume restrictions in combination with injectable anesthesia. We show using our newly defined methods for strict intranasal immunization that these methods impact the immune responses and efficacy observed when compared to vaccination methods resulting in distribution to both the upper and lower respiratory tracts. These data emphasize the importance of well-characterized immunization methods in the preclinical assessment of intranasally delivered vaccine candidates. Copyright 2018 Merck Sharp & Dohme Corp and The Authors. Published by Elsevier Ltd.. All rights reserved.