The development of a relatively simple, reliant and cost-effective animal test will greatly facilitate drug development. In this study, our goal was the establishment of a rapid, simple, sensitive and reproducible zebrafish xenograft model for anti-cancer drug screening. We optimized the conditions for the cancer cell xenograft in terms of injected cell numbers, incubation temperature and time. A range of human carcinoma cell types were stained with a fluorescent dye prior to injection into the fish larvae. Subsequent cancer cell dissemination was observed under fluorescent microscopy. Differences in injected cell numbers were reflected in the rate of dissemination from the xenograft site. Paclitaxel, known as a microtubule stabilizer, dose-dependently inhibited cancer cell dissemination in our zebrafish xenograft model. An anti-migratory drug, LY294002 (phosphatidylinositol 3-kinase inhibitor) also decreased the cancer cell dissemination. Chemical modifications to increase cancer drug pharmacokinetics, such as increased solubility (17-DMAG compared to geldanamycin) could also be assessed in our xenograft model. In addition to testing our new model using known anti-cancer drugs, we carried out further validation by screening a tagged triazine library. Two novel anti-cancer drug candidates were discovered. Therefore, our zebrafish xenograft model provides a vertebrate animal system for the rapid screening and pre-clinical testing of novel anti-cancer agents, prior to the requirement for testing in mammals. Our model system should greatly facilitate drug development for cancer therapy because of its speed, simplicity and reproducibility.