Antibiotics present the greatest threat to soil and aquatic ecosystems among the different therapeutic groups of medicines (which include prescription drugs and treatments for cancer). The strongest drugs, antibiotics, have been utilized to stop the growth of microorganisms or eradicate them. Using high-performance liquid chromatography technology with fluorescence detection, the amounts of levofloxacin and tetracycline in the wastewater from three hospitals (Medical City, Al-Kindi, and Al-Yarmouk) were determined. Levofloxacin and tetracycline were chosen in this study because they are the most important water pollutants. These antibiotic residues were separated and measured using a gradient elution technique on a reverse-phase C18