Herpesviruses are large, double-stranded DNA viruses that infect up to 100% of the population and represent a significant public health burden. Throughout their finely controlled lifecycles, herpesviruses express between 70 and 250 viral proteins with expression patterns that are characterized by a temporal cascade whereby subsequent expression of viral genes depends on the prior expression of other viral genes for successful infection. Considering their substantial protein coding capacity, the development of a method to systematically monitor viral protein levels throughout infection is not readily attainable using antibody or RNA-based approaches. Here, we present targeted mass spectrometry-based assays that comprehensively quantify viral protein levels for viruses that span the alpha-, beta-, and gamma-herpesvirus subfamilies: herpes simplex virus type I (HSV-1), human cytomegalovirus (HCMV), and Kaposi sarcoma-associated herpesvirus (KSHV), respectively. Our parallel reaction monitoring (PRM)-based assays measure 59 (HSV-1), 90 (HCMV), and 62 (KSHV) viral proteins that represent all temporal classes of gene expression as well as different components of the virion. We then applied our assays to assess the effects of clinically relevant as well as other potential antiviral agents on viral protein levels. We also showed that these assays are applicable to a wide variety of viral strains and infection models.