AbstractOur knowledge of the complex bronchoalveolar lavage fluid (BALF) proteome has increased significantly over the last decade; but still, there remain many aspects of the BALF proteome that need characterization. Current proteomic methodologies resolve proteins within limited dynamic ranges: thereby, being limited in their ability to examine important areas of the BALF proteome, such as low molecular weight, low abundance proteins. To ensure proper coverage of these proteins in the BALF proteome, a refined 2‐DE standard operation protocol is presented, highlighting important issues in sample collection, sample preparation, and 2‐D DIGE analysis. It is hoped that this will help advance the field of BALF proteomics, BALFomics, which has lagged behind similar biofluids such as plasma and serum.