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Noninvasive imaging of tumor vascularization in animal models provides an important tool for studying the biology of tumor angiogenesis as well as monitoring the effects of antiangiogenic therapies. Through the use of in vivo multispectral fluorescent imaging, we have discovered a distinct spectral signature associated with blood vessels present in fluorescent tumors in mice. This unique spectral signature allows for the tumor vasculature to be imaged and quantified without the use of vascular imaging probes. This noninvasive vascular imaging technique allows for real-time analysis of tumor vascularization, which provides a powerful and efficient tool for monitoring the effect of antiangiogenic therapies in preclinical animal models.