Xanthine oxidase (XO) is the last enzyme of purine catabolism. XO was separately purified by ammonium sulphate precipitation and affinity chromatography. The effect of some antibiotics which is commonly used in clinical on purified xanthine oxidase was determined in vitro. The name of antibiotics was gentamycin sulfate, sodium ampicillin, cefazolin sodium, chlarithromycin, rifamycin SV, clindamycin phosphate and kanamycin sulfate. XO was determined using xanthine as a substrate and IC50 values of these antibiotics exhibiting inhibition effects were found from graphs of activity (%) by plotting concentration of the antibiotics. Macrolid group of antibiotics, the effects of gentamycin sulfate and kanamycin sulfate were determinated on xanthine oxidase. Gentamycin sulfate increased xanthine oxidase enzyme activity but kanamycin sulfate caused an inhibitory effect on xanthine oxidase enzyme activity. In addition, sodium ampicillin and rifamycin SV caused activation on enzyme activity. Cefazolin sodium, chlarithromycin and clindamicin phosphate indicated inhibitory effect on xanthine oxidase enzyme activity. Especially, cefazolin sodium is the most effective inhibitor in studied antibiotics with the value of 5.4 x 10–4 mg/mL. This value is close to the other values found for XO’s classical inhibitors.