The viral infection of host cells is a dynamic and complex process characterized by multifaceted interactions and metabolic shifts between the virus and host. In order to better understand the molecular mechanisms involved in the viral life cycle—particularly replication, assembly, release, and the host cell response—researchers have turned to isotope labeling techniques. The employment of isotope labeling techniques as a precise method to track molecular changes is a common practice in the research community, with the aim of gaining deeper insights into the molecular mechanisms involved in the viral life cycle, including replication, assembly, release, and the host cell response. This review aims to provide a comprehensive summary of the current isotope labeling techniques commonly used for the analysis of virus-infected cells. It will also outline the relevant research protocols and their advantages and limitations. When employed in conjunction with high-resolution mass spectrometry, isotope labeling provides enhanced quantitative data, allowing researchers to track molecular dynamics in viruses and host cells with a high degree of precision.