In this study, a nested Reverse Transcriptase (RT) polymerase chain reaction (RT-PCR)-based assay, for detection of Newcastle Disease Virus (NDV) Ribonucleic acid (RNA) in clinical samples from experimentally infected chicks, was evaluated. The clinical samples used included, blood, tracheal, cloacal, liver, spleen, heart, lung, kidney and brain. The nested RT-PCR was performed in two amplification steps. In the first step, a pair of primers (nd and nd ) was used to amplify a 356 bp specific region in the F gene of NDV. In the second step, a nested pair of primers (nd and nd ) was employed to produce 216 bp amplification products, internal to the annealing sites of primers nd and nd . The 356 bp PCR products were amplified only from lung homogenate, cloacal and tracheal tissues, kidney, heart and brain. However, the 216 bp nested amplification was detected in all tissue samples collected from experimentally infected chicks. The nested amplification confirmed the identity of the first amplified product and increased the sensitivity of RT-PCR assay. RNA samples extracted from Infectious Bursal Disease Virus (IBDV) and Infectious Bronchitis virus (IBV) or total nucleic acid extracted from blood of non infected birds failed to demonstrate the primary or the nested PCR products. The described nested RT-PCR assay provide reliable, rapid, sensitive and specific diagnostic assay for detection of an outbreak of NDV infection among susceptible Birds.