Despite substantial improvements to media used for in vitro development of pre-implantation embryos, there is still evidence that these embryos are under stresses that alter normal cellular processes, which impacts embryo viability. Cultured embryos have altered mTOR signalling, increased ROS and mitochondrial activity compared to those developed in vivo. Amino acids (AA) are generally added to culture media in groups. However, the transporters for AAs have multiple substrates which may cause competitive inhibition of uptake of beneficial AAs when all AAs are present. Addition of individual amino acids, such as L-proline (Pro), to media used for fertilisation and culture, at concentrations like that in reproductive fluids, improves blastocyst development and hatching. Pro transporters are present throughout pre-implantation development and the presence of other substrates (eg glycine) prevents the beneficial effects of Pro. Our data show that the mechanism of action of Pro involves mTOR pathway activity, as well as reduced ROS, probably due to direct ROS scavenging by Pro, metabolism of Pro to produce the antioxidant glutathione and a reduction in mitochondrial activity. Overall, our data suggest that the quieter metabolic status of embryos cultured in the presence of only Pro can improve embryo viability and that design of media composition needs to take into consideration the impact of combinations of AAs on the action of Pro and other beneficial AAs.