A validated reverse phase high performance liquid chromatography method has been developed for the simultaneous determination of Bronopol in Lactulose Solution USP. Chromatography was carried out on a BDS Hypersil C18 (4.6 mm × 250 mm, 5 μm) using Water:Acetonitrile: Phosphate Buffer in the ratio of 94.5:5:0.5 (v/v/v) as the mobile phase at a flow rate of 1.0 mL/min and eluents were monitored at 214 nm. The average retention time of Bronopol was found to be 8.160 min. The method was validated for specificity, linearity, precision, accuracy, robustness and solution stability. The calibration curve was linear (R2 ≥0.9998) over the range of 4.0-12.0 μg/mL.