Abstract Purpose Preclinical imaging, with translational potential, lacks a standardized method for defining volumes of interest (VOIs), impacting data reproducibility. The aim of this study was to determine the interobserver variability of VOI sizes and standard uptake values (SUV mean and SUV max ) of different organs using the same [ 18 F]FDG-PET and PET/CT datasets analyzed by multiple observers. In addition, the effect of a standardized analysis approach was evaluated. Procedures In total, 12 observers (4 beginners and 8 experts) analyzed identical preclinical [ 18 F]FDG-PET-only and PET/CT datasets according to their local default image analysis protocols for multiple organs. Furthermore, a standardized protocol was defined, including detailed information on the respective VOI size and position for multiple organs, and all observers reanalyzed the PET/CT datasets following this protocol. Results Without standardization, significant differences in the SUV mean and SUV max were found among the observers. Coregistering CT images with PET images improved the comparability to a limited extent. The introduction of a standardized protocol that details the VOI size and position for multiple organs reduced interobserver variability and enhanced comparability. Conclusions The protocol offered clear guidelines and was particularly beneficial for beginners, resulting in improved comparability of SUV mean and SUV max values for various organs. The study suggested that incorporating an additional VOI template could further enhance the comparability of the findings in preclinical imaging analyses.