The objective of the proposed method is to develop and validate a specific, precise and accurate LC-MS method for the determination of 2-acetoxy ethyl acetoxy methyl ether (AEM) in Acyclovir. An isocratic separation was done by using YMC-PACK PRO C18, (150 x 4.6 mm, 3µm) column with a mobile phase composition of Buffer (0.1% Ammonia in water) : Methanol(25 :75v/v)at a flow rate of 0.4mL/min, diluent solution of 0.2% of 0.1N NaOH in Methanol were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) Acquisition mode and possitive polarity mode. The Retention time of AEM was found to be 5.344 minutes, LOD and LOQ were observed at 0.03 ppm and 0.11 ppm concentration respectively by signal to noise ratio method, linearity was found in the concentration range of 0.18-0.55 ppm with correlation coefficient of 0.996 and accuracy in the range of 97.7- 101% was obtained by performing percentage recovery studies. The % RSD was observed to be less than 10% for six replicates said to be precise. The proposed method was validated for accuracy, precision, sensitivity, and linearity successfully employed for quantitative determination of AEM in acyclovir.