Since the identification of the first virus-encoded microRNA (miRNA) in Epstein-Barr virus (EBV)-infected B cells in 2004, viral miRNAs have been found in different groups of herpesviruses. Viral miRNAs play an important role in regulating both viral and cellular gene expression. Identification and characterization of the cellular targets of viral miRNAs will not only advance our understanding of virus-cell interaction but might also reveal new strategies for the developments of antivirals. Our demonstration of the targeting of p53 upregulated modulator of apoptosis (PUMA) by an EBV-encoded viral miRNA provides one mechanism by which a viral miRNA facilitates viral replication by promoting cell survival. Using EBV miRNAs as an example, in this chapter, we detail the experimental procedures that can be used to identify and validate cellular targets of viral miRNAs.