Antibiotic resistance development is an ongoing process associated with irrational antibiotic use. WHO recommends regular surveillance programs for monitoring of antibiotic resistance. The present study is a step in this direction. A total of 124 clinical isolates of Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa were collected from different hospitals in Karachi. In vitro antimicrobial susceptibility studies were carried out by agar dilution method using newer quinolones that included Gatifloxacin and Levofloxacin. It was observed that 50% (n=30) isolates of Staphylococcus aureus were resistant to gatifloxacin. Gatifloxacin was more active against Pseudomonas aeruginosa (n=23) and showing complete susceptibility with MIC 1mg/L except for three very resistant strains that shown resistance at even higher concentrations. Escherichia coli (n=45) has shown 15.5% and Klebsiella pneumoniae (n=26) 34.61% resistance to gatifloxacin. Levofloxacin was more active against Staphylococcus aureus and Escherichia coli showing complete susceptibility at 0.5 mg /L concentration. Pseudomonas aeruginosa and Klebsiella pneumoniae were found to be resistant to Levofloxacin showing 36.36% and 23.08% resistance respectively. The study strongly recommends the adherence to the antibiotic policy and regular susceptibility testing to overcome the problem associated with antimicrobial resistance.