Background The Pediatric Preclinical Testing Program (PPTP) has been successfully used to determine the efficacy of novel agents against solid tumors by testing them within a mouse-flank in vivo model. To date, radiation therapy has not been applied to this system. We report on the feasibility and biologic outcomes of a pilot study using alveolar and embryonal rhabdomyosarcoma xeno-graft lines. Procedures We developed a high-throughput mouse-flank irradiation device that allows the safe delivery of radiotherapy in clinically relevant doses. For our pilot study, two rhabdomyosarcoma xenograft lines from the PPTP, Rh30 (alveolar) and Rh18 (embryonal) were selected. Using established methods, xenografts were implanted, grown to appropriate volumes, and were subjected to fractionated radiotherapy. Tumor response-rates, growth kinetics, and event-free survival time were measured. Results Once optimized, the rate of acute toxicity requiring early removal from study in 93 mice was only 3%. During the optimization phase, it was observed that the alveolar Rh30 xenograft line demonstrated a significantly greater radiation resistance than embryonal Rh18 in vivo. This finding was validated within the standardized 30 Gy treatment phase, resulting in overall treatment failure rates of 10% versus 60% for the embryonal versus alveolar subtype, respectively. Conclusions Our pilot study demonstrated the feasibility of our device which enables safe, clinically relevant focal radiation delivery to immunocompromised mice. It further recapitulated the expected clinical radiobiology.