Embryonic stem cell test (EST) from the European Centre for the Validation of Alternative Methods (ECVAM) evaluates the embryotoxic potential of substances and measures the half inhibition in viability of mouse embryonic stem cells (ESCs), fibroblasts (3T3 cells) and in cardiac differentiation of ESC. Previously, we established the developmental toxicity test method (termed EBT) applying area of embryoid bodies (EBs) instead of cardiac differentiation of EST. In the assessment of 15 substances for intralaboratory test and 6 substances for interlaboratory test. EB area was logarithmically decreased only in the toxic chemical with dose-dependent manner. In classification by the EBT-based prediction model reflecting decline in cell viability and EB area, toxicity for 20 chemicals showed similarity with the result of in vivo embryotoxic test. Conclusively, EBT is advanced and is a useful tool to assess and classify various embryotoxicants in a short time with less effort with accuracy.