The aim of the study is construction of a standard curve for molecular titration of the Pigeon Pox virus and uses it to quantify the virus concentrations in vaccine samples. The standard curve showed a linear relationship between cycle threshold values (Ct value) and Log10 virus titers, indicating the accuracy of this method. The amplification reactions of the Pigeon Pox vaccines samples and Fowl Pox virus exhibited specific amplification products without anomalies, as confirmed by melting curve analysis. The melting curve analysis also revealed the presence of two distinct viral strains or variants in the Pigeon Pox vaccine samples, each with a slightly different melting temperature, while the Fowl Pox virus sample contained a single viral strain or variant. The Ct values obtained from the rt-PCR analysis provided information about the amount of viral RNA present in the tested samples, with lower Ct values indicating higher concentrations of viral RNA. Additionally, the study highlights the need for further investigations about into the observed more than 100% efficiency in the standard curve to ensure result accuracy. Overall, these findings contribute to the understanding of virus concentrations in the tested vaccines and the specificity of the amplification reactions.