The potential of harnessing one’s immune system to combat cancer is well recognised, but the specific roles played by distinct immune cell populations have not been fully delineated. Animal models of human cancer have demonstrated that a specific dendritic cell (DC) subset – CD8α⁺/CD103⁺ DCs (CD141⁺ DCs in humans) can be harnessed to enhance responses to immunotherapeutic drugs, underscoring their potential as cancer vaccines to induce or boost anti-tumour immunity. DCs can be numerically and functionally impaired in some cancer patients but the integrity of CD141⁺ DCs, and hence feasibility of exploiting them for immunotherapy in human cancer is unknown. We developed a novel whole blood assay for absolute quantitation, phenotyping and functional analysis of human CD141⁺ DCs, CD1c⁺ DCs, and plasmacytoid DCs (pDCs) by flow cytometry and compared these in healthy donors and in patients with Stage III and IV metastatic melanoma. We examined DC functional properties post activation ex vivo by toll-like receptor 3 and 7/8 agonists (PolyIC and R848 respectively), and assessed CD83, CD40, the immune-inhibitory ligand PD-L1, and novel inhibitory receptor BTLA. Different DC subsets exhibit differential marker expression under steady state, and the regulation of these markers upon activation is disparate across DC subsets (eg. BTLA expression is high and remains unchanged in CD141⁺ DCs; moderate but gets downregulated in pDCs; low and remains unchanged in CD1c⁺ DCs). By contrast, the classical DC activation marker CD83 was induced in all 3 DC subsets, as were CD40 and PD-L1, albeit to a different extent. Remarkably, however, there were no significant differences in any of the tested parameters between patient and healthy control DC subsets. Given their functional integrity, targeting DCs particularly the human CD141⁺ DC subset by enhancing their numbers or function may represent viable strategies in the treatment of advanced melanoma (and other cancer) patients.