Cytometry by time of flight (CyTOF) is a flow cytometry-based technique using metal-tagged antibodies, allowing immunophenotyping. Here, we present a protocol for phenotyping mouse myeloid and lymphoid cells isolated from lung, spleen, and intraperitoneal lavages in healthy or pathologic conditions. We describe steps for antibody labeling and titration, tissue dissociations, and staining. We then detail procedures to compare staining obtained from fresh or cryopreserved tissues. For complete details on the use and execution of this protocol, please refer to Morin et al.