The study focused mainly on developing and validating the positive control from the cDNA of the gene expressing the cathepsin B3 enzyme secreted by the juvenile fluke of Fasciola gigantica in large ruminants only. The aim is to develop a positive control of the gene expressing cathepsin B3 gene from immature Fasciola gigantica flukes for prepatent detection among large ruminants. RT-LAMP optimization to detect Cathepsin B3 in marita of an infected liver showed optimal temperature and reaction time for amplifying FgCatB3 gene at 62C for 90 minutes, respectively, as confirmed in the dye, fluorescence test, gel electrophoresis, and RT-PCR. Field validation of the RT-LAMP protocol was done on swamp buffaloes, and cattle blood samples to detect the FgCatB3 gene showed a positivity rate of 27.5 % based on dye, fluorescence tests, and gel electrophoresis. Prepatent detection of fasciolos is in large ruminants can be possible using the optimized RT-LAMP that can amplify that gene expressing the cathepsin B3 enzyme, which is secreted exclusively by juvenile flukes. Blood samples can be examined for prepatent detection. Hence, strategic deworming can be implemented before the flukes reach the adult stage and obstruct the bile ducts leading to death. (Justify, Calibri 8)