Background: Assessing biomarkers to predict response is crucial for optimizing melanoma treatment with immune checkpoint inhibitors (ICIs). In this context, monitoring cytokine dynamics over time provides a rational strategy to determine whether IL-6R inhibition with sarilumab is biologically active and to identify pharmacodynamic signatures associated with clinical outcomes. Here, we focus on longitudinal serum cytokine profiling using Luminex assays to explore pharmacodynamic biomarkers for response in a phase II trial combining nivolumab (3 mg/kg), ipilimumab (1 mg/kg), the IL-6 receptor inhibitor sarilumab (150 mg), and the LAG-3 antibody relatlimab (160 mg) (INR + S) in patients with unresectable or advanced melanoma. Methods: We analyzed serum cytokines from 33 melanoma patients treated with INR + S using samples from baseline, week 4, week 6, week 12, week 18, and week 28 (final timepoint after discontinuation of sarilumab), and categorized patients according to response and toxicity. Patients were classified into PR/CR, SD, and PD groups, as well as grade 1-2 versus grade 3 or greater toxicity. Serum cytokines were profiled using Luminex assays, measuring 23 analytes. In total, 152 samples from 33 patients were analyzed. Statistical analyses were conducted using Mann-Whitney U test, paired t-test, and Kruskal-Wallis test, with the significance level set at 0.05. Results: The best overall response rate was 63.6%. Grade 3-5 irAEs occurred in 12% of patients by week 24. At baseline, no pretreatment predictive factors for either treatment response or > grade 3 irAEs were identified. Across all patients, several significant post-treatment patterns that correlated with clinical benefit were observed compared with baseline at one or more time points for IL-6, IL-6R, OSM, CXCL10, IL-4, IL-10, G-CSF, and M-CSF. Notably, PR/CR patients showed increased IL-6 and decreased IL-6R levels over time, which returned to baseline levels after discontinuation of sarilumab. When comparing baseline with week 4, progressive disease was associated with marked increases in OPN, and IL-8, whereas PR/CR patients showed decreases in IL-6R, IL-4, and G-CSF. Conclusion: Longitudinal analyses in the present study revealed increased IL-6 and decreased IL-6R levels. These dynamics likely reflect a more active mode of IL-6R blockade and may have contributed to improved clinical outcomes. The next phase of the trial is a randomized comparison of INR +/- S, and biospecimens from this randomized comparison will be analyzed to build on our findings.