The analysis of biological fluids to identify proteins that may indicate a disease setting, state and progression, is an increasingly explored field. Despite the expectatives created, there are several hurdles that must be solved to reach an extensive proteome coverage using mass spectrometry, mainly due to the complex composition of the matrices. In this regard, bile is specially challenging and yet, very attractive, as a proximal fluid that might provide valuable information for the management of liver and pancreas associated diseases. Proteins account for less than 5% of bile organic components and, although optimized protocols for protein extraction have been developed, only partial descriptions of bile proteome have been achieved. In this manuscript a new procedure is described that significantly improves protein recovery from rat bile, which reduces by a factor of six the sample amount required for a typical proteomics analysis. Moreover, the number of proteins reliably identified in a single nanoLC-MS/MS run from 1 μg protein was increased by three-fold. This procedure provides a valuable resource to dig deeper into the molecular composition of bile and open new avenues to identify new hallmarks of disease such as cholangiocarcinoma, hepatocellular carcinoma and pancreatic cancer for their better clinical management.Copyright © 2020. Published by Elsevier B.V.