Updated project metadata. Although blood-based protein biomarkers have been described for diagnosis of some cancers, detection of tumor-derived peptides/proteins in urine provides an attractive and non-invasive alternative for diagnostic or screening purposes. For instance, bladder tumor antigen (BTA) assay is FDA approved urine-based biomarker assay to detect the bladder cancer. In this study, we decided to investigate the possibility of discovering proteins as biomarkers in urine from individuals diagnosed with gastric cancer. We carried out comprehensive quantitative profiling of urine samples using tandem mass tags (TMT)-based multiplexed mass spectrometry approach. Of the 1,504 total number of proteins identified, 246 proteins were differentially expressed in a gastric cancer cohort. Ephrin A1 (EFNA1), pepsinogen A3 (PGA3) and vitronectin (VTN) were among the upregulated proteins which are known to play crucial role in the progression of gastric cancer. Notably, our analysis also revealed other overexpressed proteins including shisa family member 5 (SHISA5), mucin like 1 (MUCL1) and leukocyte cell derived chemotaxin 2 (LECT2), which had not previously been linked to gastric cancer. We decided to deploy a recently developed targeted method, SureQuant, for validating a subset of potential proteins discovered in this study. We confirmed the overexpression of EFNA1, VTN and SORT1 in an independent set of urine samples. Our study demonstrates the potential of urinary proteomics to identify promising diagnostic biomarkers of cancers in a non-invasive fashion.