Nanocrystals offer a promising strategy for enhancing the bioavailability of poorly water-soluble drugs and enabling versatile administration routes. However, their clinical translation is hindered by an incomplete understanding of their in vivo fate due to the inability to differentiate intact nanocrystals from dissolved drug molecules. This chapter introduces advances in strategies for probing nanocrystal biofate, including transmission electron microscopy, physiologically based pharmacokinetic modeling, self-fluorescent drugs, and hybrid nanocrystal technology. Hybrid nanocrystals embedded with environment-responsive probes enable real-time tracking of intact particles, revealing that nanocrystals dissolve gradually and remain intact across the epithelial membrane. Key findings highlight prolonged gastrointestinal retention after oral administration, RES-organ accumulation post-intravenous injection, follicular deposition in transdermal delivery, and slow dissolution kinetics. Quantitative particokinetic data derived from fluorescence signals demonstrate that intact nanocrystals contribute significantly to bioavailability. The integration of in vivo dissolution profiling further informs in vitro–in vivo correlation development. These insights underscore the dual biofate pathways of nanocrystals (dissolution vs. particle absorption) and emphasize the need for particle-specific tracking to optimize formulation design and clinical outcomes.