We report the development and validation of a 12 parameter immunofluorescence flow cytometry method for the sensitive determination of cell concentrations, their expression of PD-1, and PD-1 receptor occupancy. Cell subsets include CD4 + and CD8 +-T-cells, B-cells, natural killer cells, classical-, intermediate- and non-classical monocytes, and myeloid- and plasmacytoid dendritic cells. Cells were isolated from peripheral blood by density gradient centrifugation. The validation parameters included specificity, linearity, limit of quantification, precision, biological within- and between subject variations. The lower limit of quantification was 5.0% of PD-1 + cells. Samples were stable for at least 153 days of storage at −80°C. The clinical applicability of the method was demonstrated in 11 advanced cancer patients by the successful determination of immune cell concentrations, relative number of PD-1 + immune cells, and number of PD-1 molecules per immune cell. Shortly after infusion of nivolumab, receptor occupancy on CD8 +-T-cells was 98%. Similar values were found predose cycle 2, suggesting receptor occupancy remained high throughout the entire cycle.