A simple, sensitive and rapid LC- ESI-MS/MS method has been developed and validated for the trace analysis (>1 ppm level) of 2-acetoxy methyl-4-methoxy-3,5-dimethyl pyridine, a genotoxic impurity, in esomeprazole magnesium drug. The chromatographic separation was achieved on a hypersil BDS (150 x 4.6 mm, 5 µm) column using a mobile phase consisting of 5 mM ammonium acetate buffer (pH 4) and acetonitrile (60:40, v/v) at flow rate of 0.7 mL/min and elution was monitored at 305nm. The API-4000 LC-MS/MS was operated on an electrospray ionization equipped with an ESI interface operated in positive ionization (single reaction monitoring) mode and it is able to quantitate up to 0.3 ppm of 2- acetoxy methyl-4-methoxy-3,5-dimethyl pyridine. The newly developed method was validated as per ICH guidelines.