Reactivation of T cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy. However, PD-L1 immunohistochemical readout is inconsistent with patient response, which presents a clinical challenge to stratify patients. Because PD-L1 is heavily glycosylated, we developed a method to resolve this by removing the glycan moieties from cell surface antigens via enzymatic digestion, a process termed sample deglycosylation. Notably, deglycosylation significantly improves anti-PD-L1 antibody binding affinity and signal intensity, resulting in more accurate PD-L1 quantification and prediction of clinical outcome. This proposed method of PD-L1 antigen retrieval may provide a practical and timely approach to reduce false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy.