Laser-based tissue microdissection of lungs from mice infected with A. fumigatus and the subsequent MALDI Imaging MS offers the possibility to excise defined regions and transfer them to the more sensitive and deeper LC-MS/MS-based proteome analysis. In case of host-pathogen interaction between mammalian cells and pathogenic fungi like A. fumigatus this combination opens up the possibility to elucidate this biological cross-talk together with spatial Information on the invasive fungal infection. Here, we provide for the first time insights into the in vivo proteome of murine lungs infected by A. fumigatus using MALDI-IMS and laser microdissection.