RATIONALE In the manufacturing process of Trimetazidine hydrochloride (TMZ), N Nitroso trimetazidine (NTMZ) generate, final purification of active pharmaceutical ingredient (API) done in the presence of the charcoal, which has been identified as a possible source of nitrite. Secondary amine is present in TMZ, due to the favorable condition for nitrosamine & NDSRIs, NTMZ is generated in TMZ. Significant genotoxic and mutagenic effects of NDSRIs effect on human and regulatory requirement to control. METHODS This study is held for the trace level identification and quantification of NTMZ in TMZ drug substance and in drug product by using ultra-fast liquid chromatography-mass spectrometry/mass spectrometry (UFLC-ESI-MS/MS). The method was developed and validated for the NTMZ content in gradient mode using inert Sustain PFP (4.6 mm x 150 mm x 5 µ) column, a buffer containing 0.63 g/L ammonium formate in water and 1 mL formic acid as solution A and while solution B a mixture of methanol and acetonitrile in the ratio of 50:50 (v/v). Positive ionization in electrospray ionization (ESI) with MRM ( m/z 296.15 > 181.15, 296.15 > 166.05, 296.15 > 136.15) used for NTMZ quantification and identification. RESULTS The limit of detection and Quantification 0.16 ppm and 0.50 ppm respectively. The method is linear in the range of 0.5 ppm to 7.5 ppm (0.00025 ppm to 0.00375 ppm). System precision (% RSD= 4.47), Method precision (% RSD = 1.92), Intermediate precision (% RSD = 2.47), Accuracy (77.7 – 126.85 %). CONCLUSIONS To identify and quantify the trace level NTMZ in commercial drug substances and drug products, this validated analytical method was effectively applied. As the knowledge this is the first method to identify and quantify NTMZ with UPLC-ESI-MS/MS and no comparable techniques.