Abstract Background: Helicobacter pylori (H. pylori) is a bacterial carcinogen and the leading risk factor for noncardia gastric cancer (NCGC). Detecting antibodies against specific H. pylori proteins in peripheral blood can be applied to characterize infection and determine disease associations. Most studies analyzing the association between H. pylori infection and gastric cancer have focused on previously identified antigens, predominantly the virulence factor cytotoxin-associated gene A (CagA). Selecting antigens in an unbiased approach may, however, allow the identification of novel biomarkers. Methods: Using a combination of multiple spotting technique and cell-free, on-chip protein expression, we displayed the H. pylori genome (strain 26695) on high-density microarrays. Immunogenic proteins were identified by serum pool incubations and henceforth analyzed in individual samples. To test its applicability, we used sera from a multicase–control (MCC)-Spain study. Serologic responses between NCGC cases and controls were assessed by conditional logistic regression estimating ORs and 95% confidence intervals. Results: We successfully expressed 93% of the 1,440 H. pylori open reading frames in situ. Of these, 231 (17%) were found to be immunogenic. By comparing 58 NCGC cases with 58 matched controls, we confirmed a higher seroprevalence of CagA among cases (66%) than controls (31%). We further identified a potential novel marker, the Helicobacter outer membrane protein A (HopA). Conclusions: In this study, we provide evidence that our H. pylori whole-proteome microarray offers a platform for unbiased de novo identification of serologic biomarkers. Impact: Given its versatile workflow, antibody responses against other H. pylori strains and possible associations with diverse H. pylori–related outcomes can be systematically analyzed.