Background: Tumor PD-L1 expression by immunohistochemistry has limitations in describing the tumor immune microenvironment as it relates to treatment with PD-1/PD-L-1 antibodies. We developed a novel FACS based method to study the tumor immune microenvironment and used it in patients undergoing PD-1/PD-L-1 antibody treatment. Methods: Multi -parameter flow cytometry was performed on freshly harvested metastatic melanoma tumor tissue following overnight enzymatic digestion (n = 161). The gating strategy sorted tumor cells, CD4+, CD8+ and myeloid/stromal cells. CD8+ cells were further sorted for PD-1, PD-L1, CTLA-4, and HLA-DR, while CD4+ cells were sorted for FoxP3 in addition to these markers. Tumor associated macrophage and dendritic cell activation markers were also determined in samples. Overall responses were derived from investigator reported data by Response Evaluation Criteria in Solid Tumors (RECIST). Descriptive statistics for responders vs. non-responders to anti-PD-1 therapy were constructed to assess the prognostic utility of these markers. Results: TME-FACS was evaluable on 32 unique patients who were evaluable for response. A high percentage (> 35% of total CD8+ cells) of PD-1 and CTLA-4expressing CD8⁺TILs is associated with an increased response to anti-PD-1 therapy. Increased mean fluorescence intensity (MFI) of PD-1/CTLA-4 dual expression on CD8+ cells is also associated with response. Myeloid cell markers clearly stratified responders vs. non-responders to anti-PD-1 therapy. Conclusions: TME-FACS is a novel method to functionally define the immune microenvironment in melanoma. TME-FACS immunoprofiling revealed that an increased percentage of PD-1/CTLA-4 + CD8+ TIL populations in metastatic lesions correlates with responsiveness to anti-PD-1 therapies. Defined myeloid subsets can also be uncovered by TME-FACS.