Updated project metadata. Despite high clinical need, hardly any biomarker can accurately predict if patients with metastatic melanoma will respond to anti-PD-1 therapy. In this multicenter study we applied depletion and enrichment methods prior to different proteomic techniques to analyze the discovery cohort (n=56) and discovered several significantly regulated proteins as well as commonly enriched processes such as neutrophil degranulation, cell-substrate adhesion, and extracellular matrix organization. We then analyzed two independent serum cohorts (n=96; n=17) confirming significant differences between R and NR. In addition, literature-based validation revealed 30 markers overlapping with previously published signatures, and survival analysis revealed that overexpression of 17 markers correlated with lower overall survival in melanoma patients. Primary melanoma tumor cells from NR also exhibit a distinctive immunophenotype characterized by CD29, CD49e, CD91, CD105, CD151, CD157, CD248 and CD280, and the TME could represent a potential target for therapy. Ultimately, this led to a potential marker signature with 8 key markers identified in at least two independent serum cohorts: CFHR3, CRP, LRG1, LYVE1, MMRN1, S100A8, SAA2, and TIMP1.