Preclinical PET/CT is a well-established noninvasive imaging tool for studying disease development/progression and the development of novel radiotracers and pharmaceuticals for clinical applications. Despite this pivotal role, standardization of preclinical PET/CT protocols, including CT absorbed dose guidelines, is essentially nonexistent. This study (1) quantitatively assesses the variability of current preclinical PET/CT acquisition and reconstruction protocols routinely used across multiple centers and scanners; and (2) proposes acquisition and reconstruction PET/CT protocols for standardization of multicenter data, optimized for routine scanning in the preclinical PET/CT laboratory. Methods: Five different commercial preclinical PET/CT scanners in Europe and the United States were enrolled. Seven different PET/CT phantoms were used for evaluating biases on default/general scanner protocols, followed by developing standardized protocols. PET, CT, and absorbed dose biases were assessed. Results: Site default CT protocols were the following: greatest extracted Hounsfield units (HU) were 133 HU for water and −967 HU for air; significant differences in all tissue equivalent material (TEM) groups were measured. The average CT absorbed doses for mouse and rat were 72 mGy and 40 mGy, respectively. Standardized CT protocol were the following: greatest extracted HU were −77 HU for water and −990 HU for air; TEM precision improved with a reduction in variability for each tissue group. The average CT absorbed dose for mouse and rat decreased to 37 mGy and 24 mGy, respectively. Site default PET protocols were the following: uniformity was substandard in one scanner, recovery coefficients (RCs) were either over- or underestimated (maximum of 43%), standard uptake values (SUVs) were biased by a maximum of 44%. Standardized PET protocols were the following: scanner with substandard uniformity improved by 36%, RC variability decreased by 13% points, and SUV accuracy improved to 10%. Conclusion: Data revealed important quantitative biases in preclinical PET/CT and absorbed doses with default protocols. Standardized protocols showed improvements in measured PET/CT accuracy and precision with reduced CT absorbed dose across sites. Adhering to standardized protocols generates reproducible and consistent preclinical imaging datasets, thus augmenting translation of research findings to the clinic.