Molecular imaging has become an interesting and growing clinical practice and valuable research field. It permits one to understand the molecular pathways and biochemical changes associated with disease development. It also allows researchers to interrogate particular expression of key molecules that play essential role in diagnostic or therapeutic processes. Small animals remain instrumental in deciphering many biological aspects of human diseases. The implementation of modern imaging technologies in preclinical models provides excellent tools for investigating intrinsic molecular and biochemical changes associated with a given disease model. The role of micro-positron emission tomography (µPET) in this context is quite unique and has potential to prove its utilities in drug discovery and development. One of the challenges associated with this technology is selection of appropriate animal models, how far it can represent a human disorder, and how the experimental outcome is predictive of future clinical trials. Animal anaesthesia, which if not taken into consideration could have an adverse impact on data interpretation. Animal handling and monitoring protocols are key during preclinical imaging. There are also some questions regarding the performance of the µPET scanner used to acquire the data and how this information can be integrated with other modalities or correlated with histopathological finding to reveal valuable and reliable outcome. The applications of µPET imaging are numerous and have been extensively reported in the literature with special focus on cardiology, neurology and oncology.