The development of effective relief for cancer is one of the most urgent tasks of biomedicine. Despite the success of anti-tumor nanotherapeutics, low targeted delivery effectiveness remains a major limiting factor for widespread introduction of those into clinical practice. Tumor microenvironment is a complex, multicomponent system, the dynamic interaction of which with nanoparticles requires adequate analysis methods. Intravital microscopy presents a unique opportunity for in vivo assessment of drugs and body’s cells in the real-time mode. The review describes the possibilities and prospects of using intravital microscopy to study the nanotherapeutic biodistribution and delivery to tumor cells in preclinical animal models.