The lack of methodological standardization diminishes the validity of results obtained and the conclusions drawn when studying the lung microbiota. We report the validation of a complete 16S rRNA gene amplicon sequencing workflow, from patient recruitment to bioinformatics, tailored to the constrains of the pulmonary environment. We minimize the impact of contaminants and establish negative controls to track and account for them at every step. Enzymatic and mechanical homogenization combined to commercially available extraction kits allow for a fast and reliable extraction of bacterial DNA. The DNA extraction kits have a significant impact on the bacterial composition of the controls. The bacterial signatures of extracted cancerous and healthy human tissues from 5 patients are highly distinguishable from methodological controls. Our work expands our understanding of low microbial burdened environments analysis. This article is to be a starting point towards methodological standardization and the implementation of proper sampling procedures in the study of lung microbiota.