Positron emission tomography (PET) is a powerful non-invasive probe to investigate human physiology. A large number of radiotracers have been studied as imaging agents, but only a few have found clinical applications in pharmacology. A potential radiopharmaceutical is designed with very specific physiochemical characteristics, but, generally, less attention is paid to its adsorption, distribution, metabolism, and excretion properties, especially metabolism. Understanding the metabolic fate of radiopharmaceutical probes is essential for an accurate analysis and interpretation of PET measurements. The inherent inability of PET to differentiate between a parent compound and its metabolites confounds the interpretation of images and may impact the identification of the pathologically induced biochemical changes under investigation. Cytochrome P450 plays a major role in mammalian xenobiotic biotransformation and many in vitro methods are available to study and predict drug metabolism. The purpose of this review is to highlight the existing in vitro techniques available to investigate the biotransformation of xenobiotics in a fashion analogous to small molecule drug discovery. The aim is to facilitate the development and validation phases of PET tracers during preclinical evaluation. Emphasis is placed also on describing how cross species comparisons are essential in establishing appropriate translational pharmacology. Procedures of analysis (tandem liquid chromatography-mass spectrometry), typically used for studying the metabolism of drugs, are proposed as quick and accurate tools for the determination of a radiopharmaceutical's metabolic stability at the tracer level.