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The induction of teratoma in mice by the transplantation of stem cells into extra-uterine sites has been used as a read-out for cellular pluripotency since the initial description of this phenomenon in 1954. Since then, the teratoma assay has remained the assay of choice to demonstrate pluripotency, gaining prominence during the recent hype surrounding human stem cell research. However, the scientific significance of the teratoma assay has been debated due to the fact that transplanted cells are exposed to a non-physiological environment. Since many mice are used for a result that is heavily questioned, it is time to reconsider the teratoma assay from an ethical point of view. Candidate alternatives to the teratoma assay comprise the directed differentiation of pluripotent stem cells into organotypic cells, differentiation of cells in embryoid bodies, the analysis of pluripotency-associated biomarkers with high correlation to the teratoma forming potential of stem cells, predictive epigenetic footprints, or a combination of these technologies. Each of these assays is capable of addressing one or more aspects of pluripotency, however it is essential that these assays are validated to provide an accepted robust, reproducible alternative. In particular, the rapidly expanding number of human induced pluripotent stem cell lines, requires the development of simple, affordable standardized in vitro and in silico assays to reduce the number of animal experiments performed.