The swift appearance of viral pathogens and the substantial influence of viral infections require the ongoing advancement of antiviral medications. This review article offers a comprehensive analysis of in vivo and in vitro models employed for assessing the effectiveness of antiviral drugs. The text explores the essential attributes of viruses, their life cycles, and the obstacles encountered in the development of novel antiviral medications. The study emphasizes the intricacies associated with drug discovery, such as the absence of distinct targets, restricted availability of appropriate models, and the high selectivity of antiviral drugs. Additionally, it examines other cell culture systems, including Vero cells, Huh-7 cells, and other cell lines that are used for studying HCV, HBV, and influenza viruses. The significance of evaluating medication toxicity and antiviral effectiveness using cytotoxicity assays and median effective concentration (EC50) data is also highlighted. This comprehensive overview highlights the necessity for sophisticated and novel methods to improve the development of antiviral drugs and addresses the pressing global need for effective antiviral treatments.