Sensitive LC-MS/MS techniques were developed and verified for 2-Methoxy 4-Amino-5-Fluro pyramidine and L-menthyl glyoxalate contaminants in the active pharmaceutical ingredient (API) of Emtricitabine.Ultra Performance Liquid Chromatography, Acquity H-Class system, with a Quaternary Solvent manager, gradient mixer assembly, Sample Manager - F.T.N (Flow through needle) auto injector, with a column oven coupled to Xevo TQS Triple Quadrupole LC/MS/MS Mass Spectrometer, (Make Waters).Column was employed in the method was Acquity UPLC CSH Phenyl Hexyl 1.7 μm. (150mm x 2.1mm) and X-Select CSH C18 150x4.6 3.5μm mm respectively. All the weighing in the experiments was done with Sartorius balance capable of measuring with an accuracy of 0.01 mg.The method was developed and validated as per of ICH guideline. The suggested approach was robust, accurate, linear, and specific. Over the concentration range of 0.6 μg/g to 9.5 μg/g and 0.6 μg/ml to 9.4 μg/ml, the calibration curves demonstrated satisfactory linearity; the correlation coefficients were.0.999 and.0.999 in each case. The method's very low limit of quantification (LOQ) and limit of detection (LOD) were 0.3 μg/g and 0.6 μg/g, respectively.Analytical test method for determination of L-Menthyl Glyoxalate and 2-Methoxy 4-Amino-5-Fluro pyramidine in Emtricitabine by LC-MS/MS was validated for System Suitability, Identification, Specificity, Linearity, Method Precision,