Distillers Dried Grains with Solubles (DDGS) mainly contains protein, oil, fiber, and ash. DDGS could have more economic value and wider use if it could be separated into higher protein fraction and higher fiber fraction. Various ways have been explored in recent years, and two of the most effective processes are sieving and winnowing. In the present work, the optimization of three parameters of a gravity separator (side slope, eccentric shaft vibration, and the air flow rate), was conducted to separate DDGS. DDGS was firstly sieved into five size categories, then three categories and raw DDGS were further separated into light, mid-light, mid-heavy and heavy fractions using the gravity separator. By adjusting the three parameters, four combinations were tested. Nutrient analysis was measured to determine the most effective combination for DDGS fractionation. This process was found to be effective in getting substantial fractions enriched in protein and oil.