For a systemically administered therapeutic agent to reach neoplastic cells, it must enter the blood circulation, cross the vessel wall, move through the extracellular matrix and avoid getting cleared by the lymphatics. In tumours, each of these barriers is abnormal, changes with space and time, and depends on host-tumour interactions. Intravital microscopy has provided unprecedented molecular, cellular, anatomical and functional insights into these barriers and has revealed new approaches to improved detection and treatment.