Alkyl methanesulfonates have been highlighted as potential genotoxic impurities (PGIs). A sensitive LC/MS/MS method was developed and validated for the determination of Alkyl methanesulfonate impurities in Emtricitabine API (active pharmaceutical ingredient). LC/MS/MS method on Zorbax SB C18 column (150 × 4.6 mm i.d.), 3.5 μm, with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode was used. The proposed method was specific, linear, accurate, rugged and precise. The calibration curves showed good linearity over the concentration range of 0.0025 μg/ml to 0.3 μg/ml the correlation coefficient was >0.999 in each case. Method had very low limit of detection (LOD) and limit of quantification (LOQ) as 0.3 μg/g and 0.4 μg/g respectively for both the analytes. Accuracy was observed within 80%–120% for both the analytes. This method can be further extended a good quality control tool for low level quantitation of Alkyl methanesulfonate impurities in other API.