Human liver synthesizes bile; bile, containing a large number of metabolites, is transported through the canaliculi and bile ducts, and stored in the gallbladder before entering into the intestine. In the intestine, a large number of bile metabolites are reabsorbed and sent back to the liver for recirculation. Owing to close association of the bile with the gastrointestinal system, the bile metabolic profile is highly sensitive to the onset of numerous gastrointestinal disease processes. A growing number of studies suggest that hepatobiliary disease biomarkers are richly populated in human bile. These studies stress the potential of profiling the human bile metabolome for early diagnostics as well as deeper insights into gastrointestinal disease processes. Once the biomarkers are established reliably using human bile, they can be targeted in easily accessible fluids such as blood and urine or targeted in bile itself using noninvasive methods such as in vivo magnetic resonance spectroscopy. NMR spectroscopy is one of the most powerful bioanalytical tools, which promises profiling of human bile metabolome and exploring early biomarkers for hepatobiliary diseases. Comprehensive analysis of human bile using NMR spectroscopy has lead to identification and quantification of major bile metabolites. This review describes the discovery and quantitation of biomarkers of hepatobiliary diseases in human bile using NMR spectroscopy.