Recently, respiratory viral infections have witnessed substantial diagnostic evolution, with the advent of emergent pathogenic agents and the improvement of new diagnostic approaches. Acute viral respiratory infection (ARVI) commonly causes illness and fatality, particularly in pediatric patients. Adenovirus has the highest prevalence in upper and lower respiratory tract infections (RTIs) amongst respiratory pathogens of importance. The purpose of this research was to introduce two effective techniques for the detection of adenovirus respiratory infection in Human samples, cell culture, and real-time PCR. Samples from patients aged less than ten years were obtained from Baqiyatallah (as) Hospital and cultured on HEK cells after preparation. After observing the cytopathic effects of the virus, a molecular test (Real-time PCR) with the help of specific primers was done on samples taken from the cell culture. Both assays detected viruses in clinical samples, and results of cell culture and real-time PCR confirmed infection with adenovirus in samples. According to our findings, simultaneous use of tests (cell culture and PCR) based test real-time PCR is a somewhat reliable method for detecting adenovirus in clinical specimens.