There is growing interest in establishing alternative methods in place of conventional animal tests to assess the developmental and reproductive toxicity (DART) of chemicals. Gastruloids are 3D aggregates of pluripotent stem cells that spontaneously exhibit axial elongation morphogenesis similar to gastrulation. They have been explored as in vitro embryogenesis models for developmental and toxicological studies. Here, a mouse gastruloid-based assay was validated for DART assessment in accordance with the ICH S5(R3) guideline, which provides the plasma concentration data of various reference drugs in rodents, specifically Cmax and AUC for NOAEL and LOAEL. First, adverse effect concentrations of the reference drugs and their known metabolites on gastruloid development were determined based on morphological impact, namely reduced growth or aberrant elongation. Then, the NOAEL to LOAEL concentration range obtained from the gastruloid assay was compared with that in rodents to examine similarities in sensitivity between the in vitro and in vivo assays for each chemical. For 18 out of the 24 reference drugs that have both NOAEL and LOAEL information in rodents, the sensitivity of the gastruloid assay was comparable to the in vivo assay within an 8-fold concentration margin. For 7 out of the 8 additional reference drugs that have only NOAEL or LOAEL information in rodents, the gastruloid assay was in line with the in vivo data. Altogether, these results support the effectiveness of the gastruloid assay, which may be exploited as a non-animal alternative method for DART assessment.