Abstract - Newcastle disease is a highly contagious disease of poultry and causes huge economic loss in Bangladesh. In the study, the efficacy of different vaccination programs against Newcastle disease virus was performed to examine duration and level of antibody in two layer farms with different diluents. The experiment was carried out in Dinajpur district and serological test was performed in the Department of Microbiology, Hajee Mohammad Danesh Science and Technology University, Dinajpur during the period of July, 2017 to December, 2017. A total of 200 blood samples were randomly collected from different age groups of layer birds which were vaccinated with commercially available two live and one killed vaccine namely Avi ND LaSota, CEVAC NEW L and ITA-New (ND). Pre-vaccination serum antibody titers (Mean ? SD) of group A birds were 5.97?0.75. Firstly, birds were vaccinated with Avi ND LASOTA?R vaccine, and CEVAC?R NEW L vaccine. MASTERBLUE powder and ADVANCE NON-FAT were used as diluents in vaccination. After 40 days of post-vaccination, sera samples were procured and showed serum antibody titer (Mean ? SD) 7.0?0.93. In case of farm-B, before vaccination antibody titers levels against NDV were Mean ? SD 5.85?0.75. Birds of farm-B were vaccinated with Avi ND LaSota?R vaccine. After 40 days of post-vaccination, titer was Mean ? SD 6.7?0.93. Then birds were vaccinated with ITA-New (ND)?R which were killed vaccine. After 40 days of post-vaccination, the titer was Mean ? SD 8.15?0.93. MASTERBLUE powder and ADVANCE NON-FAT are suitable diluents to be used in vaccination against Newcastle disease because tap water may inactivate the vaccine due to its impurity in addition to containing higher level of chlorine and even the type of pipes or vessels used to distribute the drinking water. The results showed that the level of protection of vaccinated birds was satisfactory.