AbstractInsertional mutant libraries of microorganisms can be applied in negative depletion screens to decipher gene functions. Because of underrepresentation in colonized tissue, one major bottleneck is analysis of species that colonize hosts. To overcome this, we developed insertion pool sequencing (iPool‐Seq). iPool‐Seq allows direct analysis of colonized tissue due to high specificity for insertional mutant cassettes. Here, we describe detailed protocols for infection as well as genomic DNA extraction to study the interaction between the corn smut fungus Ustilago maydis and its host maize. In addition, we provide protocols for library preparation and bioinformatic data analysis that are applicable to any host‐microbe interaction system. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.