In vitro effects of penicillin, sulbactum, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation and homogenate and 2'.5'-ADP Seprarose 4B affinity chromotography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 11.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition , I-50 values of the antibodies were determined by plotting activity % vs. antibiotic concentration. I-50 values were 17.71 mM for penicillin, 27.38 mM for sulbactum, 28.88 mM for cefazolin, and 30.59 mM for amikacine.