Quality control systems for assays and assay components are crucial to achieving optimum standards in healthcare. In human IVF, the mouse embryo assay is commonly used to assess the quality of media and instruments. Despite its popularity, there are some well-known issues with the assay, including insensitivity to sub-optimal conditions, ethical issues due to animal use and variation in end-point interpretation between operators. Here, we detail the development of a P19 embryonic carcinoma cell-based assay with the potential to be an alternative method for quality control testing of human IVF media, and we use this assay to test varying levels of the critical energy sources pyruvate, glutamine and calcium lactate in human blastocyst media. Trademark features of the mammalian cold stress response were also incorporated as potential tools for increasing assay sensitivity. Assessed by resorufin fluorescence, P19 cells responded to changes in pyruvate and glutamine in the presence of cold stress and the mTOR inhibitor rapamycin but demonstrated natural sensitivity to calcium lactate concentrations. In addition to increasing cell sensitivity, cold stress enhanced cell growth, whilst rapamycin treatment further amplified the effects of cold stress. The assay described is robust, suitable for detecting sub-optimal culture conditions and indicates the extent to which different energy substrates affect cell growth and proliferation.