Background Xenograft mouse models represent helpful tools for preclinical studies on human tumors. For modeling the complexity of the human disease, primary tumor cells are by far superior to established cell lines. As qualified exemplary model, patients' acute lymphoblastic leukemia cells reliably engraft in mice inducing orthotopic disseminated leukemia closely resembling the disease in men. Unfortunately, disease monitoring of acute lymphoblastic leukemia in mice is hampered by lack of a suitable readout parameter. Design and Methods Patients' acute lymphoblastic leukemia cells were lentivirally transduced to express the membrane-bound form of Gaussia luciferase. In vivo imaging was established in individual patients' leukemias and extensively validated. Results Bioluminescence in vivo imaging enabled reliable and continuous follow-up of individual mice. Light emission strictly correlated to post mortem quantification of leukemic burden and revealed a logarithmic, time and cell number dependent growth pattern. Imaging conveniently quantified frequencies of leukemia initiating cells in limiting dilution transplantation assays. Upon detecting a single leukemia cell within more than 10,000 bone marrow cells, imaging enabled monitoring minimal residual disease, time to tumor re-growth and relapse. Imaging quantified therapy effects precisely and with low variances, discriminating treatment failure from partial and complete responses. Conclusions For the first time, we characterized in detail how in vivo imaging reforms preclinical studies on patient-derived tumors upon increasing monitoring resolution. In the future, in vivo imaging will enable performing precise preclinical studies on a broad range of highly demanding clinical challenges, such as treatment failure, resistance in leukemia initiating cells, minimal residual disease and relapse.