Background: Since BALF were directly collected from the tumor environment than plasma, we expected that biomarkers for the lung cancer may exist in higher concentration in BALF than in blood. Here, we developed a novel method for in-depth proteomic analysis of BALF by combining antibody-based depletion of high abundant proteins from BALF with high pH pep tide fractionation. Material and Methods; BALF samples were collected from segmental bronchus from 14 patients with lung cancers, which were centrifuged at 500 g for 5 minutes at 4°C to remove debris. Two milliliters from each of 14 patients were pooled and concentrated using Amicon filters at 5,000 g for 60 min at 4°C. Depletion was performed to remove 14 high abundant proteins Then, high pH reversed phase liquid chromatography fractionation was carried out using UltiMate 3000 HPLC. Peptides were analyzed on a high resolution Orbitrap Fusion mass spectrometer. Results: MaxQuant search resulted in the identification of 4,615 protein groups, from which 3,786 proteins were in common between lung tumors and BALF from lung cancer patients. Interestingly, 748 were specifically enriched in BALF as compared to lung tumors. We found that 28 potential biomarker proteins make up ~26% of protein concentration in BALF while ~14% in human plasma. Conclusion: We developed a novel proteomic approach that combined antibody-based depletion with high pH RPLC fractionation to maximize the BALF protein identification from lung cancer patients. Our result implied that BALF contains concentrated lung cancer biomarkers and suggests a possibility that BALF is a better source than plasma for the diagnosis of the lung cancer. We believe that this method help find novel biomarkers for the early diagnosis of the lung cancer and would facilitate the lung cancer research.