Multicomponent nucleic acid enzymes (MNAzymes) are nucleic acid enzymes composed of multiple oligonucleotide partzymes that only associate to form catalytic complexes in the presence of a target nucleic acid. Once assembled, MNAzymes cleave a separate substrate (probe) between fluorophore and quencher labels to produce a fluorescent signal indicative of the presence of the target. MNAzymes are particularly useful as tools for monitoring the accumulation of amplicons during real-time quantitative PCR (qPCR). The partzyme pairs have sensor domains that are complementary to adjacent regions in the amplicons such that their partial catalytic core domains form a complete active MNAzyme core. The probe-binding domain of the partzymes can be complementary to any one of a series of well-characterized universal probes. Since there is no need to synthesize and optimize new target-specific probes for each new target, MNAzyme qPCR provides a flexible alternative which allows target-specific interrogation with a generic readout. A series of universal probes have been designed that perform with high reliability, yielding consistent and reproducible results for any target, making the development of multiplex qPCR assays faster, cheaper, and simpler. This chapter describes a 5plex MNAzyme RT-qPCR method which simultaneously quantifies five mRNA transcripts with high efficiency and specificity using five unique universal probes, each labeled with a different fluorophore.