Ascites is a valuable source of cancer biomarkers, because it contains a variety of secreted and shed proteins from cancerous cells. Conversely, most proteomic studies on ascites have focused on ovarian cancer and provided insufficient depth for biomarker discovery. Further, no proteomic analysis of ascites from gastric cancer has been reported. To this end, we profiled the human ascites proteome to obtain a pool of biomarker candidates using comprehensive proteomic strategies. Subsequently, label-free quantitation was performed to compare the abundance of proteins between benign disease and gastric cancer patients.