A new, sensitive and rapid Ultra-Performance Liquid Chromatography (UPLC) method developed and validated for the determination of potential genotoxic impurity namely 2-(Chloromethyl)-4-methoxy-3,5-dimethylpyridine (CMDP) at trace level in omeprazole by applying the concept of Threshold of Toxicological Concern (TTC), a limit of 12.5 ppm was calculated based on the maximum daily dose of omeprazole drug substance. The UPLC method was developed and validated by using Acquity UPLC HSS C18, 1.8 (100 × 2.1 mm) column with oven temperature maintaining at 40°C. 0.01 M Phosphate buffer pH 2.5 and acetonitrile were chosen as mobile phase A and B in gradient reverse phase mode. Chromatographic parameters i.e., Flow rate: 0.20 ml/min, wave length detection: 205 nm, injection volume: 5 μl and run time: 18 min were applied for this methodology. The proposed method is specific, sensitive, accurate and precise. The established limits of Limit of Detection (LOD) and Limit of Quantification (LOQ) for this impurity are found to be 1.1 ppm and 3.3 ppm respectively. The average recovery obtained was 100.6% at 4 levels in 12 determinations for CMDP in omeprazole drug substance. This method is a good quality control tool for quantitation of CMDP at very low level in omeprazole. The experimental results are discussed in detail in this research paper.