Effect of proteolytic enzymes (trypsin, chymotrypsin and proteinase k) on biofilm formation of three S. Gallinarum strains (SG 119/ Pak, SG421/India and SG 2a 189/China) using test tube assay via CV staining method. Briefly bacteria were grown in test tubes. 30mM PBS containing 5 X MIC of trypsin, chymotrypsin and proteinase k was added in each set of test tubes respectively. CV staining method was used to determine inhibitory the effect of enzymes on biofilm. Results are replica of three independent experiment. One way ANOVA followed by post hoc Tukey test was used to analyze results in SPSS (Version 13.0). Bars with no common superscript are significantly different (p<0.05).