Pluripotent stem cells (PSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCs is cell transmission through the germline, but for human PSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles.