Exosomes mediate intracellular communication between cancer cells and the local/distant microenvironment, which promotes systemic dissemination of cancer. Here, we present a protocol for tumor-derived exosome isolation and in vivo metastasis evaluation in a mouse model. We describe steps for isolating and characterizing exosomes, establishing a metastatic mouse model, and injecting exosomes into mouse. We then detail hematoxylin and eosin staining and analysis. This protocol can be used to investigate exosome function and identify unexplored metastatic regulators related to exosome biogenesis. For complete details on the use and execution of this protocol, please refer to Lee et al. (2023).