<p>This study investigated the potential of assessing human embryo viability through the analysis of the embryo culture medium used in assisted reproductive technology (ART) via Raman spectroscopy. Two types of culture media—one for early development (medium A) and one for recovery after vitrifying/warming (medium B)—were analyzed. Our results showed that protein concentrations were higher in medium A in which embryos reached the blastocyst stage compared to those in which embryos arrested before this stage. Moreover, the pH of medium A associated with higher-grade blastocysts tended to be more acidic than that associated with lower-grade blastocysts. Conversely, in medium B, pregnancy did not occur even with high-grade blastocysts when the medium was contaminated by residual vitrifying/warming agents or had slightly elevated component concentrations due to water evaporation. Furthermore, medium B tended to be slightly alkaline with higher-grade blastocysts, in contrast to medium A. The noncontact, noninvasive, and label-free Raman spectroscopic method for assessing human embryo viability based on culture media analysis may serve as a liquid biopsy in the near future. As ART has become an important treatment for infertility, this method, which may enhance its safety and efficacy, is expected to contribute to human welfare.</p>