Alternative fuels for diesel engines are becoming important due to the decrease of petroleum reservoirs and the increase of environment pollution problems. The biodiesel is technically competitive with conventional petroleum-derived diesel fuel and requires no changes in the fuel distribution system. Injection process of biodiesel influences the atomization and dispersion of fuel in the combustion chamber. In diesel Engine different tests have been performed to improve the efficiency in cycle, power, less emission, speed, etc. There are various methods of visualizing the combustion chamber in a Diesel engine. For visualizing spray characteristics of combustion chamber in Diesel engine the window of 10mm diameter hole, transparent quartz glass materials are used, which can with-stand 1500°C temperature and pressure of about 1000 bar and engine is hand cranked for conducting the experiments. Spray characteristics of palm oil methyl ester (POME) and diesel were studied experimentally. Spray penetration and spray angle were measured in a combustion chamber of DI diesel engine by employing high definition video camera and image processing technique. The study shows the POME gives longer spray tip penetration and spray angle are smaller than those of diesel fuels. This is due to the viscosity and density of biodiesel.