Abstract: Nanocarrier drug delivery systems (NDDSs) can improve the pharmacokinetic behavior of drugs, target the delivery of active pharmaceutical ingredients to diseased areas and control drug release. In recent years, with the rapid development of nanotechnology, NDDSs have shown great potential in the field of medicine and have become a hot research topic. Precise analysis of the in vivo fate of NDDSs is key to revealing their complex in vivo processes and achieving safe and effective delivery of nanomedicines. NDDSs in vivo encompass various components such as encapsulated drugs, free drugs, nanocarriers, and nanocarrier materials, making it highly challenging to comprehensively and accurately analyze the dynamic changes of these components within the complex living system. Limited by analytical methods and techniques, there are still many challenges in the study of the in vivo fate of NDDSs, which greatly hinders their rapid development and clinical translation. This review focuses on new analytical methods and technologies as well as their applications in nano-pharmacokinetics for free drugs, encapsulated drugs, nanocarriers and nanomaterials in NDDSs. It also proposes future research directions in this field, aiming to provide some reference for relevant research on the in vivo fate of NDDSs.