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The aim of this research was to address the molecular mechanisms of methyl isocyanate genotoxicity on cell cycle regulatory proteins and to evaluate the expression of p21 protein in cultured mammalian cells. Moreover, this damage causing a cascade series of ATM, p53 and p21 proteins initiation, leads to cell cycle arrest and starts either the DNA repair mechanisms or apoptosis, but if p21 gene is get mutated due to genomic toxicity, the p21 protein is unable to express which results hindrance in cell cycle arrest mechanisms, further, cells budge to the cancerous states. For implementing the investigation, the chosen mouse cell line namely, MM55.K and NIH/3T3 were selected. Parameters of the study were Qualitative Analysis of p21 protein through Immunofluorescence, Quantitative Analysis of p21 protein through Western Blot and Kinetic of p21 protein through Flow-cytometery. This investigation was part of a major research project of Department of Research, Bhopal Memorial Hospital and Research Centre, Bhopal-462033 (M.P), India. All the investigations were carried out as per the norms of the Institutional Review Board (IRB) of the BMHRC.