Objective: The antimicrobial drugs especially Quinolones are the top most therapeutics class of drug prescribed in some country like Pakistan in primary and tertiary care hospitals and clinics. The objective of the study was to assess the irrational prescribing practice of quinolones in tertiary care setups of Karachi. Methodology: A prospective observational study was conducted for the period of 04 months from November 2019-February 2020, in Public and Private Sector Tertiary Care setups of Karachi. Data was collected from surgical, medical and emergency wards on the basis of World Health Organization prescribing pattern, to evaluate the prescriptions based on Culture sensitivity tests (CST), empirical, targeted, and/or prophylactic therapies. Results: Out of total 1000 patients 41.6% patients were from Public and 58.4% were from Private Sector Tertiary Care Hospital. 13.7% of the patients from Public and 24.8% from Private Sector Tertiary Hospitals were prescribed Quinolones after performing their Culture Sensitivity Test. However, 79.8% of the patients were prescribed Quinolones without Culture Sensitivity Test. 36.4% patients were prescribed with empirical therapy, 20.2% with De-Escalation therapy, while 43.4% were prescribed with prophylactic therapy of quinolones. Conclusion: Study concluded that some serious efforts are required to avoid the irrational prescribing practice and promote the rational prescribing practice of antibiotics particularly for Quinolones.