Levofloxacin is a third generation antibiotic belongs to a subset fluoroquinolone, extensively studied and applied in biomedical field of sciences. The main focus of presented research work was to develop an analytical methodology using UV spectrophotometer for the validation of levofloxacin in pharmaceutical injectable formulations (Leflox). Different validation parameters were performed using 0.1N HCl as diluent, for levofloxacin. The results of all these performed parameters were found within acceptable ranges, as provided by ICH guidelines. The developed methodology was linear in examined range of 3-7µg/mL with correlation coefficient (0.99997). The absorbance maxima for levofloxacin was measured at 293nm using 0.1N HCl as a blank. The precision and recovery studies were performed and results were found in close agreement with the values designed by ICH. The % RSD was found 0.00223% for precision and 0.001639-0.001641% for intermediate precision. The calculated mean percentage recovery was found to be 99.92 to 100.05%, indicated that the investigated method was established and implemented successfully for routine validation or analytical assessment of levofloxacin in injectable form.