Bile is a body fluid produced by the liver and drained by biliary ducts into the duodenum. It has two major functions: first, it contains bile acids, which are critical for the digestion of fats, and second, it is an excretory pathway for many endogenous and exogenous compounds. Proteomic analysis of bile is particularly difficult since this fluid contains high concentrations of various substances that strongly interfere with protein separation and identification techniques. Furthermore, owing to its deep location in the body, bile must be collected by surgical or endoscopic procedures. However, as was speculated for other body fluids, bile appears to be a promising sample for the discovery of disease biomarkers leaking from proximal tissues: the liver, pancreas or biliary tree. The interest in clinical proteomics was demonstrated by two studies that identified in bile potential biomarkers for two deadly and difficult to diagnose neoplasms, pancreatic cancer and cholangiocarcinoma.