Abstract: Compositions for detecting the presence of monkeypox virus in a biological sample includes a monkeypox nucleic acid forward primer; a monkeypox nucleic acid reverse primer; and a monkeypox nucleic acid probe. Methods include a multiplex assay including a three-channel panel with a single assay per channel, that includes a monkeypox assay with a reporter and a quencher, a non-variola orthopox assay with a reporter and a quencher, and a RNase P assay with a reporter and a quencher. A kit for detecting monkeypox virus nucleic acid includes the assay components including the forward and reverser primers and probes for each of the three assays in the multiplex assay.