Virus infection of host cells requires that entry into the cell results in efficient genome release leading to translation and replication. These initial steps revolving around the entry and genomic release processes are crucial for viral progeny generation. Despite the variety of receptors used by viruses to initiate entry, evidence from both enveloped and non-enveloped viral infections is highlighting the important role played by intracellular acidic compartments in the entry of many viruses. These compartments provide connecting nodes within the endocytic network, presenting multiple viral internalization pathways. Endosomal compartments employing an internal acidic pH can trigger molecular mechanisms leading to disassembly of viral particles, thus providing appropriate genome delivery. Accordingly, viruses have evolved to select optimal intracellular conditions for promoting efficient genome release, leading to propagation of the infectious agent. This review will address the implications of cellular compartment involvement in virus infectious processes, and the roles played by the viruses' own machinery, including pH sensing mechanisms and the methodologies applied for studying acid-dependent viral entry into host cells.