When using an LC-MS/MS method for approval, there are unique challenges associated with evaluating 1-Methyl-4-nitropiperazine (MNP) in rifampicin formulations. Because at certain concentrations it could cause illness, MNP, a nitrosamine contaminant, requires precise evaluation. Among the many medications used to treat and prevent tuberculosis (TB), rimpicin stands out. As a dangerous and severe disease, tuberculosis treatment and prevention are important goals for public health. Concerns about nitrosamine contamination of clinical supplies have become a global crisis, making it all the more urgent to reduce the number of drug assessments while simultaneously ensuring patient safety. In order to ensure that various products containing rifampicin had the genotoxic nitrosamine poison (MNP) present at the FDA-mandated limit level of 5.0 ppm, this review developed the LC-MS/MS method. The intricate concept of rifampicin necessitated a balanced extraction process due to its matrix and dissolvable impacts. The created technique was granted consistent endorsement from the guidelines. The following criteria were evaluated: precision, accuracy, unambiguity, limit of quantification, and limit of detection. One point where MNP contamination in Rifampicin starts is in the drug's assembly framework. The results of the small contamination study further show that two routes, rifampicin corruption and 1-amino-4-methyl-piperazine oxidation, are viable for MNP production.