Serum is a valuable body fluid to diagnose cancer as it can be accessed with minimal invasive techniques. Studying the cancer serum proteome provides valuable insights into the pathophysiology of tumor progression. Gastric adenocarcinoma is an aggressive cancer resulting in poor prognosis, mainly due to the lack of specific early diagnostic biomarkers. To this end, we used an iTRAQ-based quantitative proteomic approach to identify differentially expressed proteins in the sera of patients diagnosed with gastric cancer. Our study resulted in the identification of 643 proteins in the serum, of which 48 proteins were found to be overexpressed and 11 proteins underexpressed in gastric cancer when compared with healthy controls. We used multiple reaction monitoring assays to validate the overexpression of potential biomarkers. This catalog of serum-based biomarkers will aid in diagnosis and prognosis of gastric cancer.