Animal handling and preparation is one of the most critical aspects of in vivo NMR imaging in small animals, and involves a broad spectrum of challenges, any of which could affect data quality and reproducibility. This chapter will outline the most critical considerations in animal handling for in vivo MRI experimentation in rodent models. Highly accurate and reproducible positioning is one of the most important aspects, since sensitivity, motion and susceptibility artifacts, animal imaging throughput, and ease of data quantification are all dependent on it. A variety of devices exist today that assist in several aspects of animal handling and positioning, each with its own advantages and limitations. This chapter will detail many of the devices that are commercially available and how they have dealt with integration of RF coil technology, restraint, anesthesia, fiducial markers, warming, and physiological monitoring. The chapter will additionally detail various aspects of animal anesthesia, maintenance of core body temperature, physiological monitoring, intubation and ventilation, and systemic contrast agent administration. An increasingly important factor in running a small animal MRI laboratory, facility biosecurity, will also be reviewed.