Melanoma is an aggressive form of skin cancer that often spreads via lymphatic pathways to regional and distant sites. Melanoma-derived lymphatic small extracellular vesicles (sEVs) play a crucial role in forming a tumor-supportive environment for metastasis, or premetastatic niche, within the first tumor draining lymph node, also known as the sentinel lymph node (SLN). Therefore, analyzing the proteomic content of tumor-draining lymphatic sEVs that deliver oncogenic molecules to the SLN is important in understanding the premetastatic niche. To reveal the proteomic landscape of lymphatic sEVs we performed multidimension liquid chromatography-tandem mass spectrometry with multiplexing (18-samples) using tandem mass tag (TMT) pro labeling to profile the lymphatic sEVs proteomes obtained from afferent lymphatic channels leading to the SLN of patients with melanoma (n=6), control afferent lymphatic channels from prophylactic mastectomy (n=3) and non-cancer post-operative lymphatic fluid (n=9). Lymphatic fluid from postoperative lymphadenectomy drains served as another control to filter out non-melanoma alteration in lymph that may be related to the procedure of surgical resection and wound healing process.