Simple Summary: In the current study, we investigated changes in the levels of plant secondary metabolites (PSMs) in a rumen buffer in vitro model and identified the microbial dynamics of the rumen sample. The data suggested that the different types of PSM including luteolin, ferulic acid, caffeic acid, coumaric acid, rutin, myricetin, vitexin, kaempferol, and quercetin were decreased after 12 h of incubation in the rumen fluid (p ≤ 0.05). In contrast, the propyl gallate concentration was not significantly changed even after 24 h of incubation in rumen fluid compared to other metabolites. Microbial dynamics study showed that the Firmicutes, Bacterodetes, Actinobacteria, and Syngergistetes were the dominant phyla found in rumen fluids. Overall, data suggested that most polyphenolic compounds may degrade or reform new complex structures in the rumen. Further in vivo studies are needed to determine the PSM conversion rate and the final products from native PSM when undergoing degradation or reforming new complexes, and the effect on the microbiome. Plant secondary metabolite (PSM) degradations and feed breakdown into small particles may occur primarily in the rumen. It is possible to predict the rate and extent of feed disappearance in the rumen during incubation by different in vitro techniques, which differ based on the PSM structures, including phenolics, and flavonoids. However, PSM degradation and conversion efficiency in the rumen remains unclear. This study's objective was to evaluate the in vitro degradation of a group of PSMs in the rumen fluid, collected from Hanwoo steer samples. PSMs including rutin, vitexin, myricetin, p-coumaric acid, ferulic acid, caffeic acid, quercetin, luteolin, propyl gallate, and kaempferol were used in their pure forms at 1mg/250 mL in a rumen fluid buffer system. The mixture of selected PSMs and buffer was incubated at 39 °C for 12–72 h, and samples were collected every 12 h and analyzed by a high-performance liquid chromatography-diode array detector (HPLC-DAD) to determine the biotransformation of the polyphenolics. The results revealed that the luteolin, ferulic acid, caffeic acid, coumaric acid, rutin, myricetin, vitexin, kaempferol, and quercetin were decreased after 12 h of incubation in the rumen fluid (p ≤ 0.05) and were more than 70% decreased at 72 h. In contrast, the propyl gallate concentrations were not significantly changed after 24 h of incubation in rumen fluid compared to other metabolites. Finally, microbial dynamics study showed that the Firmicutes, Bacterodetes, Actinobacteria, and Syngergistetes were the dominant phyla found in rumen fluids. The data suggest that most polyphenolic compounds may degrade or reform new complex structures in the rumen. [ABSTRACT FROM AUTHOR]