Background: Immune checkpoint inhibitors (ICI) are now standard-of-care treatment for patients with metastatic gastric cancer (GC). To guide patient selection for ICI therapy, programmed death ligand-1 (PD-L1) biomarker expression is routinely assessed via immunohistochemistry (IHC). Regulatory approval for ICIs is granted based on PD-L1 expression status, scored using metrics such as the combined positive score (CPS). However, with an increasing number of approved ICIs, each paired with a different PD-L1 antibody IHC assay used in their respective landmark trials, there is an unmet clinical and logistical need for harmonization. We thus investigated the interchangeability between the Dako 22C3, Dako 28-8 and Ventana SP-142 assays in GC PD-L1 IHC. Methods: In this cross-sectional study, samples were obtained via biopsy or resection of gastric cancer at the National University Hospital, Singapore. We scored 362 GC samples for PD-L1 CPS, tumor proportion score (TPS) and immune cells (IC) using a multiplex immunohistochemistry/immunofluorescence technique. 344 samples were developed into a tissue microarray (TMA), while 18 samples were used as whole slides for orthogonal validation. The samples selected for whole slide analysis were obtained from GC patients treated with ICI therapy. Results: The percentage of PD-L1 positive samples at clinically relevant CPS ≥1, ≥5 and ≥10 cut-offs (Table) for the 28-8 assay were approximately two-fold higher than that of the 22C3 (CPS≥1: 70.3% vs 49.4%, p<0.001; CPS≥5: 29.1% vs 13.4%, p<0.001; CPS≥10: 13.7% vs 7.0%, p=0.004). The mean CPS score on 28-8 assay was nearly double that of the 22C3 (6.39 ±14.5 vs 3.46±8.98, p<0.001). At the clinically important CPS≥5 cut-off, there was only moderate concordance between the 22C3 and 28-8 assays. Conclusions: Our findings suggest that scoring PD-L1 CPS with the 28-8 assay may result in higher proportion of PD-L1 positivity and higher PD-L1 scores compared to assessment with the 22C3 and other assays. Clinically, this could lead to a larger number of patients eligible and approved for ICI therapy. If assays are viewed and used interchangeability, a substantial number of patients may be inaccurately denied or granted treatment with ICIs based on the assay chosen. As such, until stronger evidence of inter-assay concordance is found, we urge caution in treating the assays as equivalent.