We have previously characterized host-response to influenza infection in human primary macrophages using traditional 2-DE based proteomics and shown that actin and RIG-I/MAVS signaling components translocate onto mitochondria upon infection [40] . However, 2-DE based proteomics suffers from certain drawbacks, most importantly it shows systematic bias against certain protein groups including membrane proteins as well as very big/small proteins and proteins with extreme pIs. Also, it requires a lot of manual work and is therefore not suitable for high-throughput studies. Here, we have used unbiased highthroughput subcellular proteomics approach to analyse the host response of human primary macrophages during influenza A virus infection in a global manner. We provide evidence that the expression and/or subcellular localization of more than one thousand host proteins is affected at the early phases of infection. Up-and downregulation of proteins in all subcellular fractions reflects the dynamic interplay between these compartments and highlights the importance of subcellular proteome characterization as many of these changes cannot be seen at total proteome level.