The coexistence and coevolution of hosts with pathogens is intrinsic to our ecosystem. Pathogenic infections induce an array of changes in the hosts that are tightly linked to the progression of infection and establishment of disease. At the cellular level, this is reflected in alterations in host cell composition, organization, and ability to communicate with other cells. Thus, changes in the host proteome, metabolome, lipidome, and secretome have started to be recognized as signatures of infectious or disease states. These alterations function to either induce host defenses that counteract the infection or promote pathogen replication for spread of infection. Consequently, the discovery and characterization of these signature changes are essential for both understanding the biology of infection and identifying novel targets for therapeutic interventions. This presentation will highlight the value of advanced mass spectrometry-based proteomics for defining the dynamics of proteome organization and understanding mechanisms of cellular defense during viral infections. Examples will be given from our studies of spatial-temporal remodeling of subcellular organelles during infection. We will show how the integration of proteomics with lipidomics, live cell microscopy, mathematical modeling, and genetic knockouts has allowed us to discover a novel function for peroxisomes in the assembly of infectious particles. Additionally, the contribution of localization-dependent posttranslational modifications will be discussed, with a focus on the role of protein acetylation in cellular host defense against infection.