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Despite the possibilities of routine clinical measures and assays on readily accessible bio-samples, it is not always essential in animals to investigate the dynamics of disease longitudinally. In this regard, minimally invasive imaging methods provide powerful tools in preclinical research. They can contribute to the ethical principle of gathering as much relevant information per animal as possible. Besides, with an obvious parallel to clinical diagnostic practice, such imaging platforms are potent and valuable instruments leading to a more refined use of animals from a welfare perspective. Non-human primates comprise highly relevant species for preclinical research to enhance our understanding of disease mechanisms and/or the development of improved prophylactic or therapeutic regimen for various human diseases. In this paper, we describe parameters that critically affect the quality of integrated positron emission tomography and computed tomography (PET–CT) in non-human primates. Lessons learned are exemplified by results from imaging experimental infectious respiratory disease in macaques; specifically tuberculosis, influenza, and SARS-CoV-2 infection. We focus on the thorax and use of 18F-fluorodeoxyglucose as a PET tracer. Recommendations are provided to guide various stages of PET–CT-supported research in non-human primates, from animal selection, scan preparation, and operation, to processing and analysis of imaging data.