Background: Poor response to anti-PD1/PD-L1 remains a clinical challenge in a subgroup of patients with metastatic melanoma. Recent evidence strongly suggests that these poor responses are associated with TGF-β signaling and CD8+ T-cell excluded tumors characterized by a collagen-rich peritumoral stroma that blocks the interaction between T cells and tumor cells. In the pursuit of identifying non-invasive biomarkers associated with a T-cell excluded phenotype and predict resistance/response to immune checkpoint inhibitor therapy, we evaluated the association between blood-based biomarkers measuring type III collagen formation and cross-linking and survival outcomes in metastatic melanoma patients treated with anti-PD1 therapy. Methods: 107 patients with metastatic melanoma who started anti-PD1 monotherapy between May 2016 – March 2019 entered in a prospective real-life study (nivolumab n = 62, pembrolizumab n = 45). Type III collagen formation (PRO-C3) and type III collagen formation and cross-linking (PC3X) were measured with ELISAs in pre-treatment serum. Biomarker levels were associated to Disease Control Rate (according to RECIST v.1.1) by Mann-Whitney test and correlated to survival outcomes by Kaplan-Meier and Cox regression analyses. Results: PRO-C3 was significantly elevated in patients with progressive disease compared to the combined group of patients with complete response, partial response and stable disease (p = 0.046). High PRO-C3 and PC3X ( > 75^th percentile) prior to treatment were significantly associated with poor overall survival (PRO-C3: HR = 2.4, p = 0.008; PC3X: HR = 2.2, p = 0.019) and progression free survival (PRO-C3: HR = 1.91, p = 0.016; PC3X: HR = 1.94, p = 0.013). The median overall survival was 417 and 511 days in biomarker high patients compared to 1269 and 1269 days in biomarker low patients, for PRO-C3 and PC3X, respectively. Conclusions: Biomarkers quantified in a pre-treatment liquid biopsy reflecting excessive collagen formation and cross-linking were associated with poor response and survival outcomes in metastatic melanoma patients treated with anti-PD1 therapy. This supports an association between collagen formation and resistance to anti-PD1 therapy. Furthermore, if validated, these non-invasive collagen biomarkers may have potential for guiding patient stratification for immune checkpoint inhibitor therapy and combination therapies. Clinical trial information: NTR7015.