Introduction: One of the most prevalent cancers in Pakistan is oral sqaumous cell carcinoma (OSCC). Its absence of well-established disease prognostic factors makes community-based screening necessary to find molecular targets for higher risk and better therapy, early identification, rehabilitation, and palliative care. Recently, it was found that NOTCH 1 is a significant oncogene connected to OSCC. Objective: The aim of this study was to identify NOTCH 1 mutations in patients with active oral cancer. If these mutations are identified, it will be clear how these genes are involved in the development of oral cancer and will also help researchers identify the genetic mechanism underlying these mutations, and if all known risk factors can be prevented, how these mutations can be prevented. Methodology: 63 samples of the said carcinoma with at least 70% tumour cells were obtained, and histopathological analyses for tumour grading were done. Spin column kits were employed and DNA was extracted from the samples. That DNA was employed to amplify the NOTCH1 gene fragment, which was then isolated using an Agarose gel electrophoresis. The satisfactory amplified products were sent for commercial DNA sequencing. The acquired sequences were compared to reference sequences using bioinformatics methods for mutation analysis. Results: Among 63 patients in the study, approximately 64.9% had poor to moderately differentiated oral squamous cell carcinoma, and approximately 53.1% had well differentiated disease. Notch mutations were found in F1-6 and F2-13 samples with missense mutations. F1-6 had an exon mutation and F2-13 had an intron mutation. Conclusion: Notch 1 mutations in the ligand binding area may serve as functional prognostic markers, and that domain may be employed as a novel therapeutic molecule in oral squamous cell carcinoma.