Update information. Anastomotic leakage (AL), one of the most severe complications in rectal surgery, is often diagnosed late because of the low specificity of clinical symptoms and limitations of current clinical investigations. Identification of patients with early AL remains challenging. Here, we explored the protein expression profiles of AL patients to provide potential biomarkers to predict AL in patients who undergo surgery for rectal cancer. We screened differentially expressed proteins (DEPs) in drainage fluid from AL and non-AL patients using a tandem mass tag (TMT) method. A total of 248 DEPs, including 98 upregulated and 150 downregulated proteins, were identified between AL and non-AL groups.