The addition of L-proline to embryo culture medium improves development of preimplantation embryos in vitro. Throughout preimplantation development, embryos are capable of L-proline uptake, although the rate of uptake is greatest during the zygote, 2-cell, and blastocyst stages. Unlike other amino acids, the concentration of L-proline in embryos has not been studied. We hypothesized that embryos developed in vitro in the absence of L-proline would contain less L-proline than those developed in the presence of L-proline. Thus, the aim of this this project was to use liquid chromatography and mass spectrometry to quantify L-proline in embryos that were developed in vivo and in vitro in the absence and presence of L-proline. It was found that in vivo developed 2-cell embryos contained significantly more L-proline than in vivo developed oocytes, zygotes, and 4-cell embryos. In addition, oocytes, zygotes, and 2-cell embryos were found to contain more L-proline when cultured with 400 µM L-proline, compared to those that were developed in vivo or cultured in the absence of L-proline. Comparatively, 4-cell embryos that had been cultured in the absence and in the presence of 400 µM L-proline contained the same amount of L-proline, which was significantly greater than that of in vivo developed 4-cell embryos, suggesting that L-proline may be produced endogenously. The elevated amount of L-proline in 2-cell and 4-cell stage embryos corresponds with the time at which L-proline improves development during in vitro embryo culture. Further knowledge of the regulation of the proline metabolic cycle and uptake in embryos is required to understand the mechanisms underlying the changing amounts of L-proline described in this study.