Gentamicin sulfate is a potent broad spectrum aminoglycoside antibiotic which is used against Gram-positive and Gram-negative bacteria. A simple, isocratic HPLC method for separation, identification and determination of gentamicin and parabens (methylparaben and propylparaben) was developed and validated. To our knowledge there is no report about simultaneous determination of those three analytes in pharmaceutical products. The optimum chromatographic conditions were achieved on CN column with a mobile phase consisting of 0.15% triethylamine in 10 mM KH2PO4 aqueous solution (final pH 3.0 adjusted with H3PO4) and methanol in the ratio 70:30 (v/v), providing selective quantification of analytes within 5 min. The method was successfully validated according to ICH guidelines acceptance criteria in terms of selectivity, linearity, accuracy, precision and robustness. The linearity of the method was proved in defined concentrations ranges for gentamicin (0.32–1.04 mg mL−1), methylparaben (0.0072–0.0234 mg mL−1) and propylparaben (0.0008–0.0026 mg mL−1). Relative standard deviations calculated for all analytes in precision testing were <2% (analysis repeatability) and <3% (intermediate precision). Recovery values were between 98.87% and 101.67%. Chromatographic parameters are not significantly influenced by small variations of column temperature, pH and molarity of KH2PO4. Finally, the method was successfully applied for quantitative determination of gentamicin and parabens in commercially available solution for injection. Proposed HPLC method is found to be promising in terms of simplicity, analysis times and non-use of derivatization and ion-pair agents.