A high accuracy method for quantitative expression proteomics was developed for the identification of putative markers in gastric adenocarcinoma. Firstly, gastric adenocarcinoma and nonmalignant epithelial cells were obtained through laser capture microdissection (LCM), which were proteolysis and then prefractionated by 1D SDS-PAGE. The post-digestion ¹⁸O/¹⁶O labeling method followed by Nano-HPLC-MS/MS were conducted to identify the differentially expressed proteins between gastric adenocaicinoma and nonmalignant gastric mucosa. A total of 78 differential proteins were identified, among these proteins, 42 proteins are over-expressed in gastric adenocarcinoma tissues and 36 proteins are down-expressed. Some of these differentially expressed proteins (moesin, periostin, annexin A2, annexin A4) were further confirmed by Western blot analysis. The quantitative proteomic protocol of ¹⁸O stable isotope labeling coupled with LCM was an effectively alternate technique for complicated proteins such as gastric cancer.