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-coatedascorbic acid, nicotinic acid, eastern herb cocktail, charcoal extract and garlic sources (lyophilized andextract) were added to rumen culture fluid at 1.25% of substrate (100% timothy) volume at 24 hincubation. pH, total gas, ammonia, VFAs were measured. Ascorbic acid increased total gas productionindicating fermentation level. All zinc compounds significantly decreased (p<0.05) although zinc sulfateincreased propionate of volatile fatty acid (p<0.05). In following experiment, ascorbic acid, oil-coatedascorbic 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 andVFAs production but ascorbic acid enhanced total gas production. Ascorbic acid increased fermentationregardless of supplement concentration although excessive zinc sulfate decreased fermentation. Theseresults suggest that optimal level of trace compounds might affect ruminal fermentation in ruminant.