Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is a tumorigenic rhadinovirus that is associated with all forms of Kaposi's sarcoma. Current serological detection of KSHV is based on enzyme-linked immunosorbent or immunofluorescence assays that suffer from a variety of problems, including the lack of defined standards for test comparison. While KSHV is the only known human rhadinovirus, two lineages of KSHV-like rhadinoviruses are found in Old World primates: the RV1 lineage includes KSHV and retroperitoneal fibromatosis herpesvirus (RFHV) in macaques, and the RV2 lineage includes RRV and MneRV2 from different macaque species. To develop animal models of KSHV-associated diseases, we developed quantitative multiplex bead-based serological assays to detect antibodies against rhadinovirus antigens. Proteins from KSHV (RV1) and MneRV2 (RV2) virions were coupled to spectrally distinct fluorescent beads and used in Luminex flow cytometry-based assays to detect immune responses in macaques. Both assays showed large dynamic ranges with high levels of seroreactivity to both KSHV and MneRV2 proteins. A large set of macaque serum samples from the Washington National Primate Research Center was screened, and most of the samples (82%) were positive in both assays, consistent with the high level of RV1-RV2 coinfection detected by PCR. The macaque sera showed broad, variable, and unique serological responses to the different viral antigens, allowing an initial seroprevalence to be determined for the macaque viruses. The Luminex assays offer a novel multiplexed approach to assess rhadinovirus infection patterns in both humans and nonhuman primates. This will help advance our understanding of rhadinovirus biology and associated host immunological responses.