Objective: This study aims to validate the feasibility of utilizing flagellin in DNA vaccine formulations by successfully cloning and expressing the fliC gene from Salmonella typhimurium into non-pathogenic bacterial hosts such as Escherichia coli, paving the way for the development of DNA vaccines. Design: The study design is "Synergistic Integration of In-Silico Analysis and Molecular Cloning Techniques." Location and Duration: Conducted at the Pathology Laboratory, Department of Biotechnology, Institute of Industrial Biotechnology, GC University Lahore, the study spanned from January 2021 to January 2023. Methodology: Leveraging reverse vaccinology, the entire proteome sequence of Salmonella typhimurium underwent rigorous analysis, spotlighting flagellin among 205 antigenic candidates. Subsequent molecular cloning facilitated the integration of the flagellin gene (fliC) from Salmonella typhimurium into E. coli DH5α strains, utilizing the pJET1.2/blunt cloning vector. Further advancement included successful subcloning into E. coli BL21 via the expression vector pET22b(+). Results: The triumphant cloning and expression of flagellin in non- pathogenic bacterial hosts underscored its potential as a formidable vaccine candidate against typhoid fever. This milestone not only validated the plausibility of utilizing flagellin in DNA vaccine formulations but also delineated promising avenues for future vaccine development strategies. Conclusion: This study illuminates the efficacy of amalgamating biotechnological tools, such as reverse vaccinology and molecular cloning, to pinpoint and express vaccine candidates. The successful cloning and expression of flagellin epitomize its promise as a constituent of DNA vaccines targeting typhoid fever, offering a promising solution to the constraints of existing vaccines.