AbstractReceptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total‐drug‐bound RO assay format directly assesses mAb binding to cell surface targets using anti‐drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total‐drug‐bound RO assay format for RG7769, a bi‐specific P329GLALA containing mAb targeting PD‐1 and TIM3 on T cells. In its fit‐for‐purpose validated version, this RO assay has been used in the Phase‐I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint‐inhibitor (CPI) naïve patients and anti‐PD‐1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre‐dosing was roughly twofold lower in patients recently having undergone anti‐PD‐1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti‐PD‐1 treatment.