Positron emission tomography (PET) has become an important tool in preclinical research, offering insights into molecular and physiological processes with 3-dimensional in vivo imaging at around 1 mm spatial resolution. This chapter highlights the applications of PET across diverse fields of preclinical research, emphasizing its unique role in drug development, cancer research, neurological studies, and cardiovascular investigations. In drug development, PET facilitates the evaluation of target engagement and pharmacokinetics, enabling the precise characterization of drug behavior in living systems. Within the domain of cancer research, PET has advanced our understanding of tumor metabolism and supported the monitoring of immunotherapy efficacy, paving the way for theranostic approaches. In neurological research, PET is instrumental in studying brain metabolism, neurotransmitter systems, neuroinflammation, and pathological protein aggregation, which are central to brain diseases such as neurodegenerative diseases. Similarly, in cardiovascular studies, PET provides valuable insights into cardiac metabolism and atherosclerotic plaque characterization, aiding in the early detection and management of cardiovascular diseases. By combining robust quantitative capabilities with high sensitivity, PET continues to enhance our understanding of disease mechanisms, accelerating the translation of preclinical discoveries into clinical advancements. This chapter reviews the current state of preclinical PET applications and highlights emerging trends and future perspectives.