Different concentration of 12 antimicrobial agents were tested by mixing into instant milk powder. Microbiological test kit for detection of antibiotic residues with tube diffusion method using Bacillus stearothermophilus var. calidolactis NIZO were employed. Compare minimum concentration of each antimicrobial agent found 100% by the prepared test kit (K2) with Maximum residue limit (MRL) and minimum concentration of that antibiotic found 100% by Delvotest-P and ADM. K2 was prepared by mixing B.stearothermophilus var. calidolactis NIZO obtained from culturing in agar media at 65 ํC for 18-20 hours into complex media. The amount of organism in test kit was equal to 10x10x10x10x10x10x10x10 CFU/ml. The capability of test kit was tested by using instant milk powder mixed with different concentration of 12 antimicrobial agents (spiked milk). The experiment showed that K2 was better in detection of beta-lactams than other antimicrobial agents. Minimum concentration of Ampicillin, Cloxacillin, Penicillin, and Cephapirin found 100% were 40, 20, 5 and 40 ppb respectively which are close to MRL value of each antibiotic. K2 could not detect Chloramphenicol with 625-40,000 ppb concentration. Minimum concentration of Streptomycin, Gentamicin, Kanamycin, Erythromycin and Oxytetracycline found 100% were 1,500, 480, 7,500, 400, and 120 ppb respectively. 400,000 ppb Subfamethazine and 400,000 ppb Subfathiazole were found 50, 33%. The concentration found 100% of these antimicrobial agents were close to those found 100% by Delvotest-P and ADM. The minimum concentration of Sulfamethazine and Sulfathiazole found 100% by K2 which added Trimethoprin 0.05 microg./ml were 500 and 250 ppb. The minimum concentration of Sulfamethazine and Sulfathiazole found 100% by K2 which added Trimethoprim 0.1 was 250 ppb. The concentration of different kinds of antibiotic found by K2 which kept at 7 ํC for 0-4 months was not changed more than four fold from the first finding and the time of reading extended not more than 30 minutes.