Abstract The traditional approach to feeding ruminant is based on the needs of protein and energy to enhance production and productivity. It is crucial to note the protein degradability in feed as microbial rumen needs ammonia from rumen degradable protein (RDP), while ruminant needs bypass protein also known as rumen undegradable protein (RUP) alongside microbial crude protein. Through the use of an in vitro technique, the recent study aimed to determine the optimal level of protein, energy, and RDP:RUP ratio in the diet of beef cattle by observing VFA partial production, protozoa population, and methane gas emissions. Various dietary treatments were formulated (based on dry matter content) comprised three protein levels (12%, 14%, and 16%), two energy levels (65% and 70%), and three RDP:RUP ratio levels(55:45 for low RDP, 60:40 for medium RDP, and 65:35 for high RDP). The study followed 3x3x2 factorial randomized block design with three replications. The dietary treatments were incubated with buffered rumen fluid. This study showed acetic, propionic, and butyric acid production were influenced by protein, energy, RD:RUP ratio (P<0.05). Meanwhile, protozoa population and methane gas emission were reduce by dietary protein, energy, and RDP:RUP ratio. In summary, dietary protein, energy, and RDP:RUP ratio impacts the production of VFA, protozoa population, and methane gas emissions. The diet comprising 14% dietary protein, 70% energy, and 60%:40% of RDP: RUP optimizes the production of VFA, while simultaneously reducing protozoa population and methane gas emissions.