Barley (Hordeum vulgare) belongs to the Poaceae family. This study compared the polyphenol and flavonoid levels of unfermented and fermented barley sprouts using spectrophotometric assays. The findings indicated that fermentation greatly boosted the flavonoid content but caused only a slight increase in the polyphenol content. However, this does not imply that fermentation has no effects whatsoever on the polyphenol content of barley sprouts. This was due to the fact that some flavonoids cannot be detected by the wavelength used to calculate the overall polyphenol concentration. Both samples were subjected to high-performance liquid chromatography analysis and detected the flavonoids lutonarin, saponarin, isoorientin, isovitexin, and tricin—all of which have bioactive properties—most notably known for their antioxidant activity. These results augment the ongoing phytochemical profiling research and can possibly valorize the already thriving barley industry.