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Background: Mycoplasma genitalium is a gram negative, parasitic pathogenic bacterium, usually transmitted sexually into human and frequently causing urethritis in men and women as well as cervicitis and pelvic inflammation in women. This is an extremely small selfreplicating entity whose genome has been sequenced. This genome sequencing is advantageous in understanding pathogenesis and identifying therapeutic targets. In this study different bioinformatics tools and databases were adopted to analyze the functions of different hypothetical proteins from M. genitalium G37. Methodology: A total of 75 hypothetical proteins (HPs) were retrieved from KEGG database, while CDD-BLAST, Pfam, and InterProScan servers were used for conserved domains analysis. After that, those HPs were broadly analyzed for physicochemical properties, subcellular localization, GO annotation, and virulence factors. Results: Based on best score, hypothetical protein MG_476 was selected for homology modelling which produced a fairly good quality 3D model. The active site within MG_476 was predicted using CASTp server that helps to explore the surface features of the protein. Other approaches include the use of NetCTL, IEDB, Bcepred, and ABCpred servers to predict the location of B and T cell epitopes. Among the CD8+ T cell epitopes tested, ILQIIMFIL scored highest (0.23718) in terms of immunogenicity. Conclusion: Moreover, this analysis recommended MG_476 as a non-homologous protein and the data generated in this study may facilitate the experimental designing of novel drug and vaccines against M. genitalium. [ABSTRACT FROM AUTHOR]