BACKGROUND: Extracellular vesicles (EVs) are crucial mediators in brain metastasis (BM), facilitating pre-metastatic niche formation and metastatic progression. However, tracking their distribution and interactions in vivo remains challenging. OBJECTIVE: To develop and validate a method for labeling BM-derived EVs using superparamagnetic iron oxide nanoparticles (SPIONs) that enables their visualization and tracking through magnetic resonance imaging (MRI). METHODS: Three SPION variants with different coatings and sizes were evaluated using two patient-derived BM cell lines. The labeled EVs were characterized using transmission electron microscopy (TEM), colorimetric iron assays, dynamic light scattering and nanoparticle tracking analysis (NTA) in Nanospacer devices. The functionality and visualization of SPION-labeled EVs were assessed in fetal rat brain organoids (FRBOs) using Prussian blue staining, TEM, and MRI. Detection sensitivity was determined using agar phantoms, and in vivo tracking was validated through intramuscular injections in mice. RESULTS: Uncoated 5 nm SPIONs demonstrated superior labeling efficiency, successfully marking over 90% of cells within 24 hours without significantly affecting cell growth. These SPIONs were effectively incorporated into BM-derived EVs while maintaining their original size distribution. The labeled EVs were successfully internalized by FRBOs and could be visualized using multiple imaging modalities. Agar phantom studies revealed significant changes in T2 and T2* relaxation times, which was further confirmed through in vivo MRI following intramuscular injections. CONCLUSION: This study establishes a reliable protocol for labeling BM-derived EVs with SPIONs, enabling their visualization across various biological contexts, from subcellular to tissue levels. This proposed model facilitates a valuable tool for spatially tracking BM-EVs in vivo, identifying specific target cells, and investigating their functional role in metastatic progression.