Abstract: Proteases, the group of enzyme with significant commercial value, was isolated from proteolytic bacteria available in gut of tiger shrimp (Penaeus monodon). The isolation of bacteria from the gut of the collected shrimp was performed by serial dilution and plating method. Six bacterial isolates were screened out on the basis of their formation of zone of casein hydrolysis on skim milk agar. Among the six isolates, three isolates (S1, S3 and S5) were found gram positive and the rest three isolates (S2, S4 and S6) were found gram negative. Protease activity of the isolates was determined both qualitatively and quantitatively. In qualitative plate assay, isolate S1 exhibited the largest clear zone (30±1.13 mm) in skim milk agar and isolate S5 exhibited the lowest (18±1.41 mm). Quantitative protease assay was performed by using azo-casein as substrate. Protease activity of isolates S1, S2, S3, S4, S5 and S6 were found 49.75±2.13, 60.50±1.97, 66.25±2.41, 56.25±1.69, 59.25±1.32 and 52.75±2.21 U mL-1, respectively following incubation at 37°C in aerobic condition for 24 h. The effect of pH and NaCl concentration on the growth and protease production of the isolates were also studied by assaying protease activity at different pH range and NaCl concentrations. The isolates exhibited maximum protease production at varying pH and NaCl concentrations. The data showed potentiality of the bacterial isolates to be a useful source of industrial protease. Finally, the isolates were also tested against six standard antibiotics (Tetracycline, Nitrofurantoin, Erythromycin, Penicillin, Ciprofloxacin and Doxycycline) to observe their antibiotic sensitivity and the isolates S5 and S6 were found resistant to all of the six antibiotics and isolates S3 and S4 were found sensitive to all of the antibiotics.