Objective: To establish a reliable, convenient and high resolution method for bile proteomic analysis. Methods: Gallbladder bile samples were drawn from 3 gallbladder cancer patients and 3 gallstone patients for protein extraction and purification, and then the protein samples were identified and quantified using a high-resolution proteomic method named isobaric tags for relative and absolute quantification (iTRAQ), and then were analyzed by bioinformatics methods. Results: After concentration and integrity test, all of the extracted protein samples met the concentration and integrity requirements of the iTRAQ experiment. Following identification, a total of 1 323 proteins were detected, which was remarkably higher than that by traditional methods. In bile from gallbladder cancer patients compared with that from gallstone patients, 173 proteins were significantly up-regulated and 345 proteins were significantly down-regulated (fold change>1.5, P<0.05). Conclusion: A reliable method for bile protein isolation, purification, identification and analysis is established, which has the ability to detect many more proteins and greater potential to find key proteins associated with specific diseases compared with traditional methods, and may lay a foundation for proteomic analysis of bile and other body fluids in the future.