A new, specific, and robust normal phase liquid chromatographic (NP-LC) method for the determination of structurally similar impurities (isomers) of Vortioxetine hydrobromide has been developed and validated. An excellent chromatographic resolution between the structurally similar impurities (isomers) and Vortioxetine hydrobromide was achieved on Chiralpak-ADH (250 x 4.6 mm ID), 5 μ column. The mobile phase used of n-hexane, ethanol, diethylamine, and trifluoroacetic acid in the ratio (75:25:0.05:0.05, v/v/v/v) and the flow rate was maintained at 1.0 mLmin-1. UV detection was carried out at 235 nm. The resolution between all positional isomers and Vortioxetine hydrobromide was found not less than 2. An effect of column oven temperature on the resolution was also studied and found to be ≥ 2.0. Three structurally similar impurities (Isomer-1, Isomer-2, and Isomer-3) have been detected in the test sample of Vortioxetine hydrobromide by advanced NP-LC method. Further, these isomeric impurities were characterized by mass spectrometry, 1H NMR and FT-IR spectral data. The developed method was validated as per ICH guidelines and found to be specific, robust, and selective. The developed NP-LC method was successfully applied to the analysis of drug substance and drug product samples of Vortioxetine hydrobromide.