This study is conducted to screen compounds affecting ruminal fermentation under in vitro incubation. Saponin, chitosan, metformin, zinc acetate, zinc chloride, zinc oxide, zinc sulfate, ascorbic acid, oil-coated ascorbic acid, nicotinic acid, eastern herb cocktail, charcoal extract and garlic sources (lyophilized and extract) were added to rumen culture fluid at 1.25% of substrate (100% timothy) volume at 24 h incubation. pH, total gas, ammonia, VFAs were measured. Ascorbic acid increased total gas production indicating fermentation level. All zinc compounds significantly decreased (p<0.05) although zinc sulfate increased propionate of volatile fatty acid (p<0.05). In following experiment, ascorbic acid, oil-coated ascorbic acid, garlic lyophilized, herb cocktail and zinc sulfate were added to rumen culture fluid at 2.5% of substrate with 3, 6, 12 and 24 h incubation. Zinc sulfate decreased both ruminal fermentation and VFAs production but ascorbic acid enhanced total gas production. Ascorbic acid increased fermentation regardless of supplement concentration although excessive zinc sulfate decreased fermentation. These results suggest that optimal level of trace compounds might affect ruminal fermentation in ruminant.