The evaluation of developmental toxicity requires new methods that provide an alternative to animal use. In this study, we evaluated the effects of thalidomide exposure [upon nanosecond pulsed electric field (nsPEF) treatment] on the embryonic development of, teratogenic effects in, and gene expression levels in medaka embryos, to determine whether the adverse effects of thalidomide can be detected using medaka. Incorporation of thalidomide into medaka embryos led to malformations in individuals. The results obtained by microinjecting thalidomide into zebrafish embryos could be reproduced by medaka embryo electroporation. Furthermore, the teratogenic mechanism of thalidomide was confirmed at the genetic level. Medaka eggs, in addition to zebrafish, can be used to assess the effects of thalidomide. As the nsPEF method can efficiently incorporate of test chemicals into medaka eggs, it is suggested to have great potential for use as a high-throughput animal alternative evaluation system.