The blood supply of solid tumours affects the outcome of treatment via its influence on the microenvironment of tumour cells and drug delivery. In addition, tumour blood vessels are an important target for cancer therapy. Intravital microscopy of tumours growing in ‘window chambers’ in animal models provides a means of directly investigating tumour angiogenesis and vascular response to treatment, in terms of both the morphology of blood vessel networks and the function of individual vessels. These techniques allow repeated measurements of the same tumour. Recently, multi-photon fluorescence microscopy techniques have been applied to these model systems to obtain 3D images of the tumour vasculature, whilst simultaneously avoiding some of the problems associated with the use of conventional fluorescence microscopy in living tissues. Here, we review the current status of this work and provide some examples of its use for studying the dynamics of tumour angiogenesis and vascular function.