This study describes the development and validation of an in-house sandwich ELISA (Exotest) to detect and quantify exosomes in plasma from melanoma patients. Exosomes are small vesicles secreted by tumor cells and are involved in malignant progression. The Exotest uses antibodies against CD63 and caveolin-1, two proteins expressed on exosomes, to capture and quantify exosomes. The assay was validated using exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. The Exotest showed high sensitivity and specificity in detecting exosomes, with a sensitivity of 69% for caveolin-1+ exosomes compared to 43% for CD63+ exosomes. Plasma levels of exosomes expressing CD63 and caveolin-1 were significantly higher in melanoma patients compared to healthy donors. The study also found that the amount of plasma exosomes correlated with tumor size in the SCID mice model. The Exotest was further tested on unfractioned plasma samples, demonstrating its potential for clinical use. The results suggest that the Exotest may be a useful tool for detecting and quantifying exosomes in melanoma patients, potentially as a biomarker for cancer screening and follow-up.This study describes the development and validation of an in-house sandwich ELISA (Exotest) to detect and quantify exosomes in plasma from melanoma patients. Exosomes are small vesicles secreted by tumor cells and are involved in malignant progression. The Exotest uses antibodies against CD63 and caveolin-1, two proteins expressed on exosomes, to capture and quantify exosomes. The assay was validated using exosomes purified from human tumor cell culture supernatants and plasma from SCID mice engrafted with human melanoma. The Exotest showed high sensitivity and specificity in detecting exosomes, with a sensitivity of 69% for caveolin-1+ exosomes compared to 43% for CD63+ exosomes. Plasma levels of exosomes expressing CD63 and caveolin-1 were significantly higher in melanoma patients compared to healthy donors. The study also found that the amount of plasma exosomes correlated with tumor size in the SCID mice model. The Exotest was further tested on unfractioned plasma samples, demonstrating its potential for clinical use. The results suggest that the Exotest may be a useful tool for detecting and quantifying exosomes in melanoma patients, potentially as a biomarker for cancer screening and follow-up.