In vivo fluorine‐19 MRI using F‐based tracer media has shown utility and versatility for a wide range of biomedical uses, particularly immune and stem cell detection, as well as biosensing. As with many advanced MRI acquisition techniques, the sensitivity and limit of detection (LOD) in vivo is a key consideration for a successful study outcome. In this review, we analyze the primary factors that limit cell LOD. The achievable sensitivity is strongly dependent on the specific composition of tracer, cell type of interest, cell activity, data acquisition and reconstruction methods, and MRI hardware design. Recent innovations in molecular 19F tracer design and image acquisition–reconstruction methods have achieved significant leaps in 19F MRI sensitivity, and integration of these new materials and methods into studies can result in > 10‐fold improvement in LOD. These developments will help unlock the full potential of clinical 19F MRI for biomedical applications.