In this article, we define systems biology of virus entry in mammalian cells as the discipline that combines several approaches to comprehensively understand the collective physical behaviour of virus entry routes, and to understand the coordinated operation of the functional modules and molecular machineries that lead to this physical behaviour. Clearly, these are extremely ambitious aims, but recent developments in different life science disciplines slowly allow us to set them as realistic, although very distant, goals. Besides classical approaches to obtain high-resolution information of the molecules, particles and machines involved, we require approaches that can monitor collective behaviour of many molecules, particles and machines simultaneously, in order to reveal design principles of the systems as a whole. Here we will discuss approaches that fall in the latter category, namely time-lapse imaging and single-particle tracking (SPT) combined with computational analysis and modelling, and genome-wide RNA interference approaches to reveal the host components required for virus entry. These techniques should in the future allow us to assign host genes to the systems’ functions and characteristics, and allow emergence-driven, in silico assembly of networks that include interactions with increasing hierarchy (molecules–multiprotein complexes–vesicles and organelles), and kinetics and subcellular spatiality, in order to allow realistic simulations of virus entry in real time.