Recently, various immuno-PET tracers based on monoclonal antibodies (mAbs), engineered scaffold proteins, and peptides were developed to target either programmed cell death protein 1 (PD-1) or programmed cell death ligand 1 (PD-L1), showing promise in assessment of immune checkpoints. We sought to develop an immunotherapeutic agent based PET probe that enables real-time assessment of PD-L1 expression and evaluation of antibody drug biodistribution to select eligible candidates for anti-PD-1/PD-L1 immunotherapies. KNO35, a 79.6 kDa size anti-PD-L1 domain antibody under analysis in clinical trials, was used to develop the immuno-PET probe, Zr-89-Df-KNO35. Immuno-PET studies were performed to monitor PD-Ll levels in nude mice bearing LN229 xenografts with positive expression for PD-Li, and to evaluate the whole-body biodistribution in healthy nonhuman primates (NHPs). LN229 xenografts were markedly visualized from 24 h after injection of Zr-89-Df-KNO35, with elevated accumulation persisting for up to 120 h. Tumor radioactivity was notably reduced in the presence of excess KNO35. Mouse ex vivo biodistribution studies performed at 24 and 120 h revealed tumor-to-muscle ratios as high as 5.64 +/- 0.65 and 7.70 +/- 1.37, respectively. In the NHP model, PET imaging demonstrated low background. The liver and kidney showed moderate accumulation with the highest SUVmean value of 1.15 +/- 0.15 and 2.13 +/- 0.10 at 72 h, respectively. The spleen, lymph nodes, and salivary glands were also slightly visualized. In conclusion, Zr-89-Df-KNO35, a novel anti-PD-Ll domain antibody-based probe, shows the feasibility of noninvasive in vivo evaluation of PD-Ll expression. This work further provides a template for immunotherapeutic agent based imaging to evaluate human PD-L1 expression and to augment our understanding of therapeutic agent biodistribution, leading to better therapeutic strategies in the future.