Plasmids containing the Epstein-Barr virus (EBV) latent origin of replication, OriP, are stably maintained in human cells expressing the viral EBNA-1 protein. This stable maintenance is owing to the ability of EBNA-1 to activate DNA replication from OriP and to facilitate the segregation of the plasmids during cell division. Methods for quantifying the replication and stable maintenance of EBV-based plasmids in human cells are presented here, as is a reconstituted segregation system in yeast that enables the segregation activity of EBNA1 to be measured independently from its replication activity.