Here, we present an in vivo drug screening protocol using a zebrafish model of metastasis for the identification of anti-metastatic drugs. A tamoxifen-controllableTwist1a-ERT2transgenic zebrafish line was established to serve as a platform for the identification. By crossingTwist1a-ERT2withxmrk(a homolog of hyperactive form of the epidermal growth factor receptor) transgenic zebrafish, which develop hepatocellular carcinoma, approximately 80% of the double transgenic zebrafish show spontaneous cell dissemination of mCherry-labeled hepatocytes from the liver to the entire abdomen and tail regions in five days, through induction of epithelial to mesenchymal transition (EMT). This rapid and high-frequency induction of cell dissemination makes it possible to perform an in vivo drug screen for the identification of anti-metastatic drugs targeting metastatic dissemination of cancer cells. The protocol evaluates the suppressor effect of a test drug on metastasis in five days, by comparing the frequencies of the fish showing abdominal and distant dissemination patterns in the test drug–treated group with those in the vehicle-treated group. Our study previously identified that adrenosterone, an inhibitor for hydroxysteroid (11-beta) dehydrogenase 1 (HSD11β1), has a suppressor effect on cell dissemination in the model. Furthermore, we validated that a pharmacologic and genetic inhibition of HSD11β1 suppressed metastatic dissemination of highly metastatic human cell lines in a zebrafish xenotransplantation model. Taken together, this protocol opens new routes for the identification of anti-metastatic drugs.Graphical overviewTimingDay 0: Zebrafish spawningDay 8: Primary tumor inductionDay 11: Chemical treatmentDay 11.5: Metastatic dissemination induction in the presence of a test chemicalDay 16: Data analysis