The influence of urine and glucose added urine on the in vitro activities of various antibiotics was assessed by the microbroth dilution method in this study. Sixteen beta-lactam susceptible Escherichia coli isolates were used. The minimum inhibitory concentrations (MICs) of ampicillin, ampicillin+sulbactam, cephalothin, cefuroxime, ceftriaxone, amikacin, ciprofloxacin, sulfamethoxazole and trimethoprim were performed parallel in Mueller Hinton broth, human urine and glucose added (440 mg/dl glucose) urine by the microbroth dilution method. MICs of nearly all antibiotics were higher in urine and glucose added urine than MICs in broth. MIC90 against ampicillin was 8 fold higher in urine and 32 fold higher in glucose added urine than MIC90 in broth. MIC90s against ceftriaxone, ciprofloxacin and trimethoprim in g-urine were only 2 fold higher than MIC90 in urine. Equal MIC90 in g-urine and urine were obtained for ampicillin-sulbactam, cephalothin, cefuroxime and sulphamethoxazole. Two fold smaller MIC90 in g-urine in comparison with MIC90 in urine were found for amikacin. The results of statistical analysis were significant (p<0.05) for all antibiotics except ciprofloxacin, sulphamethoxazole and trimethoprim for broth-urine comparison. For broth-g-urine the results were significant for all antibiotics except sulphomethoxazole and trimethoprim. The results were found unsignificant for urine -g-urine comparison. This study demonstrated that MICs of antibiotics are influenced by the human urine and glucose added urine. MICs of some antibiotics used in the treatment of urinary tract infections may be overestimated by the standard antibiotic testing methods.