In this study, Alkaline Phosphatase (EC 22.214.171.124, ALP) which has a critical role in phosphate metabolism was purified via bi- ochemical methods and the effects of some veterinary drugs on purified milk ALP were determined on account of the fact that using as a pasteurization indicator in milk. Enzyme was homogenized with different organic solvents, ammonium sulfate precipitation, dialysis and following Sephadex G-100 gel filtration chromatography. The enzyme product yielded bands, app- roximately 85 and 185-190 kDa on SDS-PAGE except casein fractions, while a single band, approximately 170-190 kDa interval. ALP is the most important enzyme in dairy industry as the check for the presence of residual ALP activity in milk is a good indication of proper pasteurization. Heat treatment of milk for adequate pasteurization, which kills pathogenic microorga- nisms, give rise to inactivate ALP. It seems as if milk is pasteurized before not to reach pasteurization temperature, in case of decreasing ALP activity notably by the veterinary drugs. Therefore, in vitro inhibitory effects of some drugs (furosemide, atropine sulfate, toldimfos sodium and levamisol/synonim: (-)- tetramisol HCI) were investigated in this study. While atropine sulfate didin’t effect the enzyme activity, the IC50 values of drugs which have inhibitory effect were obtained 45.48 μM, 2.19 and 126.315 mM for tetramisol, furosemide and toldimfos sodium, respectively. When compared to Levamisol, which is a po- tent inhibitor of the enzyme, the inhibitory effects of the drugs on the enzyme activity are weak.