Introduction: Impurity analysis is an important step in the quality control of pharmaceutical ingredients and the final product. Impurities can arise from drug synthesis or excipients and even at small concentrations may affect product efficacy and safety. In this work, two methods using highperformance liquid chromatography (HPLC) were developed and validated for the evaluation of besifloxacin and its impurity synthesis, with isocratic elution and another with gradient elution. Method: The analysis by HPLC in isocratic elution mode was performed using a cyano column maintained at 25 °C. The mobile phase was composed of 0.5% triethylamine (pH 3.0): acetonitrile (88:12 v/v) eluted at a flow rate of 1.0 mL/min with detection at 330 nm. The gradient elution method was carried out with the same column and mobile phase components only modifying the rate between organic and aqueous phase during analysis. The procedures have been validated according to internationally accepted guidelines, observing results within acceptable limits. Results: The methods presented were found to be linear in the 140 to 260 μg/ mL range for besifloxacin and 0.3 to 2.3 μg/ mL for an impurity named A. The limits of detection and quantification were, respectively, 0.07 and 0.3 μg/ mL for impurity A, with a 20 μL injection volume. The precision achieved for all analyses performed provided RSD inter-day equal to 6.47 and 6.36% for impurity A with isocratic elution and gradient, respectively. The accuracy was higher than 99% and robustness exhibited satisfactory results. In the isocratic method, an analysis time of 25 min and 15 min was obtained for the gradient. For impurity A, the number of theoretical plates in the isocratic mode was about 5000 while in the gradient mode it was about 45000, hence, it made the column more efficient by changing the mobile phase composition during elution. In besifloxacin raw material and in the pharmaceutical product used in this study, other related impurities were present but impurity A was searched for and not detected. Conclusion: The proposed methods can be applied for the quantitative determination of impurities in the analysis of the besifloxacin raw material, as well as in ophthalmic suspension of the drug, considering the quantitation limit.